Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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GLP-1 COMPOUNDS
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Patent Application No.
60/793,707, filed on April 20, 2006.
BACKGROUND
Glucagon-like peptide 1 (GLP-1) and the related peptide glucagon are produced
via differential processing of proglucagon and have opposing biological
activities.
Proglucagon itself is produced in a-cells of the pancreas and in the
enteroendocrine L-
cells, which are located primarily in the distal small intestine and colon. In
the pancreas,
glucagon is selectively cleaved from proglucagon. In
the intestine, in contrast,
proglucagon is processed to form GLP-1 and glucagon-like peptide 2 (GLP-2),
which
correspond to amino acid residues 78-107 and 126-158 of proglucagon,
respectively (see,
e.g., Irwin and Wong, 1995, MoL Endocrinol 9:267-277 and Bell et al., 1983,
Nature
304:368-371). By convention, the numbering of the amino acids of GLP-1 is
based on
the GLP-1 (1-37) formed from cleavage of proglucagon. ..The biologically
active forms
are generated from further processing of this peptide, which yields GLP-1 (7-
37)-OH and
GLP-1 (7-36)-NH2. The first amino acid of these processed peptides is His7.
Both GLP-
1 (7-37)-OH (or simply GLP-1 (7-37)) and GLP-1 (7-36)-NH2 have the same
activities.
For convenience, the term "GLP-1", is used to refer to both of these forms.
Glucagon is secreted from the a-cells of the pancreas in response to low blood
glucose, with the main target organ for glucagon being the liver. Glucagon
stimulates
glycogen breakdown and inhibits glycogen biosynthesis. It also inhibits fatty
acid
synthesis, but enhances gluconeogenesis. The net result of these actions is to
significantly increase the release of glucose from the liver. GLP-1, in
contrast, lowers
glucagon secretion, while stimulating insulin secretion, glucose uptake and
cyclic-AMP
(cAMP) formation in response to absorption of nutrients by the gut. Various
clinical data
provide evidence of these activities. The administration of GLP-1, for
example, in poorly
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controlled type 2 diabetics normalized their fasting blood glucose levels
(see, e.g.,
Gutniak, et al., 1992, New Eng. J Med. 326:1316-1322).
GLP-1 has a number of other important activities. For instance, GLP-1 also
inhibits gastric motility and gastric secretion (see, e.g., Tolessa, 1998, J.
Clin. Invest.
102:764-774). This effect, sometimes referred to as the ileal brake effect,
results in a lag
phase in the availability of nutrients, thus significantly reducing the need
for rapid insulin
response.
Studies also indicate that GLP-1 can promote cell differentiation and
replication,
which in turn aids in the preservation of pancreatic islet cells and an
increase in 13-cell
mass (See, e.g., Andreasen et al., 1994, Digestion 55:221-228; Wang, et al.,
1997, 1
Clin. Invest. 99:2883-2889; Mojsov, 1992, Int. J. Pep. Prot. Res. 40:333-343;
and Xu et
al., 1999, Diabetes 48:2270-2276). Evidence also indicates that GLP-1 can
increase
satiety and decrease food intake (see, e.g., Toft-Nielsen et al., 1999,
Diabetes Care
22:1137-1143; Flint et al., 1998, J. Clin. Invest. 101:515-520; Gutswiller et
al., 1999 Gut
44:81-86).
Other research indicates that GLP-1 induces 13-cell-specific gene expression,
including GLUT-1 transporter, insulin receptor and hexokinase-1 (see, e.g.,
Perfetti and
Merkel, 2000, Eur. J. Endocrinol. 143:717-725). Such induction could reverse
glucose
intolerance often associated with aging.
Because GLP-1 plays a key role in regulating metabolic homeostasis, it is an
attractive target for treating a variety of metabolic disorders, including
diabetes, obesity
and metabolic syndrome. Current treatments for diabetes include insulin
injection and
administration of sulfonylureas, metformin and TZDs. These approaches,
however, have
significant shortcomings. Insulin injections, for instance, require
complicated dosing
considerations, and treatment with sulfonylureas often becomes ineffective
over time,
metformin can induce hypoglycemia and TZDs have side effects such as body
weight
gain and edema. Potential advantages of GLP-1 therapy include: 1) increased
safety
because insulin secretion is dependent on hyperglycemia, 2) suppression of
glucagon
secretion which in turn suppresses excessive glucose output, and 3) slowing of
gastric
emptying, which in turn slows nutrient absorption and prevents sudden glucose
increases.
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A key hurdle for effective treatment with GLP-1, however, has been the very
short half-life of the peptide, which typically is only a few minutes (see,
e.g., Hoist, 1994,
Gastroenterology 107:1848-1855). Various analogs have been developed with the
goal
of extending the half-life of the molecule. Some of these, however, have
significant
gastrointestinal side effects, including vomiting and nausea (see, e.g.,
Agerso et al., 2002,
Diabetologia 45:195-202).
Accordingly, there thus remains a need for improved molecules that have GLP-1
type activity, for use in the treatment of various metabolic diseases such as
diabetes,
obesity, irritable bowel syndrome and metabolic syndrome.
SUMMARY
GLP-1 compounds that comprise GLP-1 analogs and have an activity of GLP-1
(e.g., insulinotropic activity) are disclosed herein. Methods for treating a
variety of
diseases by administering an effective amount of the compositions are also
provided.
Such methods can be used to treat, for example, diabetes, impaired glucose
tolerance,
insulin resistance, various lipid disorders, obesity, cardiovascular diseases
and bone
disorders.
Some of the GLP-1 compounds that are provided herein, for example, comprise a
GLP-1 analog that comprises the amino acid sequence of formula I (SEQ ID NO:
5):
Xaa7-Gly-Xaa9-Xaa 10-Xaa -Xaa12-Xaa13-Xaa 14-Xaa 5-Xaa 16-Xaa 1 7-Xaa1 8_Xaa
19-
Xaa20-Xaa2i-Xaa22-Cys-Xaa24-Xaa25-Xaa26-Xaar_Xaa28-Xaa29-Xaa30Aaa31-
Xaa32-Xaa33_Xaa34_Gly_Xaa36_Xaar-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-C(0)-R
(Formula I, SEQ ID NO: 5)
wherein,
R1 is OR2 or NR2R3;
R2 and R3 are independently hydrogen or (Ci-C8)alkyl;
Xaa at position 7 is: L-histidine,
desamino-histidine, 2-amino-
histidine, 3-hydroxy- histidine, homohistidine, a-fluoromethyl-histidine or a-
methyl-histidine;
Xaa at position 9 is Glu, Asp, or Lys;
Xaa at position 10 is Gly or His;
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Xaa at position 11 is Thr, Ala, Gly, Ser, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 12 is: His, Trp, Phe, or Tyr;
Xaa at position 13 is Thr or Gly;
Xaa at position 14 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 15 is Asp or Glu;
Xaa at position 16 is Val, Ala, Gly, Ser, Thr, Leu, Ile, Tyr, Glu, Asp, Trp,
or Lys;
Xaa at position 17 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 18 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, Trp, Tyr,
Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-
Homoglutamic acid, or homoglutamic acid;
Xaa at position 19 is Tyr, Phe, Trp, Glu, Asp, Gln, Lys, Homolysine,
Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or
homoglutamic acid;
Xaa at position 20 is Leu, Ala, Gly, Ser, Thr, Ile, Val, Glu, Asp, Met, Trp,
Tyr,
Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, or
homoglutamic acid;
Xaa at position 21 is Glu, Asp, Lys, Homolysine, Ornithine, 4-carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic
acid;
Xaa at position 22 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, Lys,
Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
or homoglutamic acid;
Xaa at position 24 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Arg, Glu, Asp, Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-
Homoglutamic acid, or homoglutamic acid;
Xaa at position 25 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, Lys,
Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
or homoglutamic acid;
Xaa at position 26 is Lys, Homolysine, Arg, Gln, Glu, Asp, His, Ornithine, 4-
carboxy-phenylalanine, beta-glutamic acid, or homoglutamic acid;
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Xaa at position 27 is Leu, Glu, Asp, Lys, Homolysine, Ornithine, 4-carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic
acid;
Xaa at position 28 is Phe, Trp, Asp, Glu, Lys, Homolysine, Ornithine, 4-
carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic
acid;
Xaa at position 29 is Ile, Leu, Val, Ala, Phe, Asp, Glu, Lys, Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
or homoglutamic acid;
Xaa at position 30 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, Lys,
Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
or homoglutamic acid;
Xaa at position 31 is Trp, Phe, Tyr, Glu, Asp, or Lys;
Xaa at position 32 is Leu, Gly, Ala, Ser, Thr, Ile, Val, Glu, Asp, or Lys;
Xaa33 is: Val or Lys;
Xaa34 is: Lys or Asn;
Xaa36 is: Arg or Gly;
Xaa37 is: Gly or Pro;
wherein the compound has a GLP-1 activity. In a particular aspect, the GLP-1
analog has
the amino acid sequence of SEQ ID NO: 7 or 8 with no more than 5 conservative
amino
acid substitutions, provided that the conservative amino acid substitutions
are not at
amino acid Xaa8, Xaa23, or any of Xaa38 to Xaa45. In another particular
aspect, the GLP-1
analog has the amino acid sequence of any of SEQ ID NO: 7 or 8.
Other GLP-1 compounds as provided herein comprise a GLP-1 analog that
comprises the amino acid sequence of formula II (SEQ ID NO: 9)
Xaa7-Xaa8-Xaa9-Xaa1 0-Xaa1 -Xaa 2-Xaa1 3-Xaa1 4-Xaa1 5-Xaa1 6-Xaa1 7-Xaa1
8_Xaa19-
Xaa20-Xaa2I-Xaa22- Xaa23-Xaa24-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29-Xaa30-Xaa31-
Xaa32-Xaa33_Xaa34Aaa35..Xaa36_Xaa37-Cys-Ser-Gly_G1y-C(0)-Ri (Formula II,
SEQ ID NO: 9)
wherein,
R1 is OR2 or NR2R3;
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R2 and R3 are independently hydrogen or (Ci-C8)alkyl;
Xaa at position 7 is: L-histidine, D-histidine, desamino-histidine, 2-amino-
histidine, 3-hydroxy-histidine, homohistidine, a-fluoromethyl-histidine or a-
methyl-histidine;
Xaa at position 8 is Gly, bAla (2-aminopropionic acid), 1-amino-
cylcopentanecarboxylic acid, Aib (2-aminoisobutryic acid) or an alpha-alpha-
disubstituted amino acid;
Xaa at position 9 is Glu, Asp, or Lys;
Xaa at position 10 is Gly or His;
Xaa at position 11 is Thr, Ala, Gly, Ser, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 12 is: His, Trp, Phe, or Tyr;
Xaa at position 13 is Thr or Gly;
Xaa at position 14 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 15 is Asp or Glu;
Xaa at position 16 is Val, Ala, Gly, Ser, Thr, Leu, Ile, Tyr, Glu, Asp, Trp,
or Lys;
Xaa at position 17 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 18 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, Trp, Tyr,
Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-
Homoglutamic acid, or homoglutamic acid;
Xaa at position 19 is Tyr, Phe, Trp, Glu, Asp, Gln, Lys, Homolysine,
Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or
homoglutamic acid;
Xaa at position 20 is Leu, Ala, Gly, Ser, Thr, Ile, Val, Glu, Asp, Met, Trp,
Tyr,
Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, or
homoglutamic acid;
Xaa at position 21 is Glu, Asp, Lys, Homolysine, Ornithine, 4-carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic
acid;
Xaa at position 22 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, Lys,
Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
or homoglutamic acid;
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Xaa at position 23 is Gin, Asn, Arg, Glu, Asp, Lys, Ornithine, 4-carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic
acid;
Xaa at position 24 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Arg, Glu, Asp, Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-
Homoglutamic acid, or homoglutamic acid;
Xaa at position 25 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, Lys,
Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
or homoglutamic acid;
Xaa at position 26 is Lys, Homolysine, Arg, Gin, Glu, Asp, His, Ornithine, 4-
carboxy-phenylalanine, beta-glutamic acid, or homoglutamic acid;
Xaa at position 27 is Leu, Glu, Asp, Lys, Homolysine, Ornithine, 4-carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic
acid;
Xaa at position 28 is Phe, Trp, Asp, Glu, Lys, Homolysine, Ornithine, 4-
carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic
acid;
Xaa at position 29 is Ile, Leu, Val, Ala, Phe, Asp, Glu, Lys, Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homogiutamic
acid,
or homoglutamic acid;
Xaa at position 30 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, Lys,
Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
or homoglutamic acid;
Xaa at position 31 is Trp, Phe, Tyr, Glu, Asp, or Lys;
Xaa at position 32 is Leu, Gly, Ala, Ser, Thr, Ile, Val, Glu, Asp, or Lys;
Xaa at position 33 is Val, Gly, Ala, Ser, Thr, Leu, Ile, Glu, Asp, or Lys;
Xaa at position 34 is Asn, Lys, Arg, Glu, Asp, or His;
Xaa at position 35 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 36 is Gly, Arg, Lys, Glu, Asp, or His;
Xaa at position 37 is Pro, Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, or
Lys;
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wherein the compound has a GLP-1 activity. In a particular aspect, the GLP-1
analog
has the amino acid sequence of SEQ ID NO: 11 or 12 with no more than 5
conservative
amino acid substitutions, provided that the conservative amino acid
substitutions are not
at amino acid Xaa8 or any of Xaa38 to Xaa41. In another particular aspect, the
GLP-1
analog has the amino acid sequence of SEQ ID NO: 11 or 12.
Still other GLP-1 compounds as provided herein comprise a GLP-1 analog that
comprises the amino acid sequence of formula III (SEQ ID NO: 13)
Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa1 2-Xaa13-Xaa14-Xaal 5-Xaa1 6-Xaa17-Xaai 8_Xaa1
9-
Xaa20-Xaa2I-Xaa22- Xaa23-Xaa24-Xaa25-Xaa26-Xaa27..Xaa28-Xaa29-Xaa30.Xaa31-
Xaa32-Xaa33_Xaa34_Xaa35_Xaa36_Gly-Xaa38_Xaa39_Xaa40_Xaa41- C(0)-R1 (Formula
III, SEQ ID NO: 13)
wherein,
R1 is OR2 or NR2R3;
R2 and R3 are independently hydrogen or (CI -C8)alkyl;
Xaa at position 7 is: L-histidine, D-histidine, desamino-histidine, 2-amino-
histidine, 3-hydroxy-histidine, homohistidine, a-fluoromethyl-histidine or a-
methyl-histidine;
Xaa at position 8 is Gly, bAla (2-aminopropionic acid), 1-amino-
cylcopentanecarboxylic acid, Aib (2-aminoisobutryic acid) or an alpha-alpha-
disubstituted amino acid;
Xaa at position 9 is Glu, Asp, or Lys;
Xaa at position 10 is Gly or His;
Xaa at position 11 is Thr, Ala, Gly, Ser, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 12 is His, Trp, Phe, or Tyr;
Xaa at position 13 is Thr or Gly;
Xaa at position 14 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 15 is Asp or Glu;
Xaa at position 16 is Val, Ala, Gly, Ser, Thr, Leu, Ile, Tyr, Glu, Asp, Trp,
or Lys;
Xaa at position 17 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
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Xaa at position 18 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, Trp, Tyr,
Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-
Homoglutamic acid, or homoglutamic acid;
Xaa at position 19 is Tyr, Phe, Trp, Glu, Asp, Gln, Lys, Homolysine,
Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or
homoglutamic acid;
Xaa at position 20 is Leu, Ala, Gly, Ser, Thr, Ile, Val, Glu, Asp, Met, Trp,
Tyr,
Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, or
homoglutamic acid;
Xaa at position 21 is Glu, Asp, Lys, Homolysine, Ornithine, 4-carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic
acid;
Xaa at position 22 is Aib (2-aminoisobutyric acid), 1-amino-
cylcopentanecarboxylic acid, an alpha-alpha-disubstituted amino acid, or Aad
(2-
amino adipic acid);
Xaa at position 23 is Gln, Asn, Arg, Glu, Asp, Lys, Ornithine, 4-carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic
acid;
Xaa at position 24 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Arg, Glu, Asp, Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-
Homoglutamic acid, or homoglutamic acid;
Xaa at position 25 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, Lys,
Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
or homoglutamic acid;
Xaa at position 26 is Lys, Homolysine, Arg, Gln, Glu, Asp, His, Ornithine, 4-
carboxy-phenylalanine, beta-glutamic acid, or homoglutamic acid;
Xaa at position 27 is Leu, Glu, Asp, Lys, Homolysine, Ornithine, 4-carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic
acid;
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Xaa at position 28 is Phe, Trp, Asp, Glu, Lys, Homolysine, Ornithine, 4-
carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic
acid;
Xaa at position 29 is Ile, Leu, Val, Ala, Phe, Asp, Glu, Lys, Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
or homoglutamic acid;
Xaa at position 30 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, Lys,
Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
or homoglutamic acid;
Xaa at position 31 is Trp, Phe, Tyr, Glu, Asp, or Lys;
Xaa at position 32 is Leu, Gly, Ala, Ser, Thr, Ile, Val, Glu, Asp, or Lys;
Xaa at position 33 is Val, Gly, Ala, Ser, Thr, Leu, Ile, Glu, Asp, or Lys;
Xaa at position 34 is Asn, Lys, Arg, Glu, Asp, or His;
Xaa at position 35 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 36 is Gly, Arg, Lys, Glu, Asp, or His;
Xaa at position 38 is Cys, Gly, or is omitted;
Xaa at position 39 is Ala, Gly, Ser, Cys, or is omitted;
Xaa at position 40 is Gly or is omitted;
Xaa at position 41 is Gly or is omitted;
provided that when the amino acid at position 38, 39, 40, or 41 is omitted,
then each
amino acid downstream of that amino acid is also omitted, and wherein the
compound
has a GLP-1 activity. In a particular aspect, the GLP-1 analog has the amino
acid
sequence of any of SEQ ID NO: 16 to 28 with no more than 5 conservative amino
acid
substitutions, provided that the conservative amino acid substitutions are not
at amino
acid Xaa8, Xaa22, or any of Xaa38to Xaa.41. In another particular aspect, the
GLP-1 analog
has the amino acid sequence of any of SEQ ID NO: 16 to 28.
Certain GLP-1 compounds as provided herein comprise a GLP-1 analog that
comprises the amino acid sequence of Formula IV (SEQ ID NO: 29)
Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Xaa1 5-Xaa16-Xaa17-Xaa18-Xaa19-
Xaa20-Xaa21-Xaa22-Xaa23-Xaa24-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29-Xaa30-Xaa31-
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Xaa32-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-Xaa38-Xaa39-Xaa40-Xaa41C(0)-R1
(Formula IV, SEQ ID NO: 29)
wherein,
R1 is OR2 or NR2R3;
R2 and R3 are independently hydrogen or (C -C8)alkyl;
Xaa at position 7 is: L-histidine, D-histidine, desamino-histidine, 2-amino-
histidine, 3-hydroxy-histidine, homohistidine, a-fluoromethyl-histidine or a-
methyl-histidine;
Xaa at position 8 is Gly, bAla (2-aminopropionic acid), 1-amino-
cylcopentanecarboxylic acid, 2-aminoisobutryic acid or an alpha-alpha-
disubstituted
amino acid;
Xaa at position 9 is Glu, Asp, or Lys;
Xaa at position 10 is Gly or His;
Xaa at position 11 is Thr, Ala, Gly, Ser, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 12 is: His, Trp, Phe, or Tyr;
Xaa at position 13 is Thr or Gly;
Xaa at position 14 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 15 is Asp or Glu;
Xaa at position 16 is Val, Ala, Gly, Ser, Thr, Leu, Ile, Tyr, Glu, Asp, Trp,
or Lys;
Xaa at position 17 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 18 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, Trp, Tyr,
Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-
Homoglutamic acid, or homoglutamic acid;
Xaa at position 19 is Tyr, Phe, Trp, Glu, Asp, Gin, Lys, Homolysine,
Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, alpha,
gamma-diaminobutryic acid, or homoglutamic acid;
Xaa at position 20 is Leu, Ala, Gly, Ser, Thr, Ile, Val, Glu, Asp, Met, Trp,
Tyr,
Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid,
alpha,
gamma-diaminobutryic acid, or homoglutamic acid;
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Xaa at position 21 is Glu, Asp, Lys, Homolysine, Ornithine, 4-carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or alpha, gamma-
diaminobutryic acid, homoglutamic acid;
Xaa at position 22 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, Lys,
Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
alpha, gamma-diaminobutryic acid, or homoglutamic acid;
Xaa at position 23 is Gln, Asn, Arg, Glu, Asp, Lys, Ornithine, 4-carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, alpha, gamma-
diaminobutryic acid, or homoglutamic acid;
Xaa at position 24 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Arg, Glu, Asp, Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-
Homoglutamic acid, alpha, gamma-diaminobutryic acid, or homoglutamic acid;
Xaa at position 25 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, Lys,
Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
alpha, gamma-diaminobutryic acid, or homoglutamic acid;
Xaa at position 26 is Lys, Homolysine, Arg, Gin, Glu, Asp, His, Ornithine, 4-
carboxy-phenylalanine, beta-glutamic acid, alpha, gamma-diaminobutryic acid,
or
homoglutamic acid;
Xaa at position 27 is Leu, Glu, Asp, Lys, Homolysine, Ornithine, 4-carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, alpha, gamma-
diaminobutryic acid, or homoglutamic acid;
Xaa at position 28 is Phe, Trp, Asp, Glu, Lys, Homolysine, Ornithine, 4-
carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, alpha, gamma-
diaminobutryic acid, or homoglutamic acid;
Xaa at position 29 is Ile, Leu, Val, Ala, Phe, Asp, Glu, Lys, Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
alpha, gamma-diaminobutryic acid, or homoglutamic acid;
Xaa at position 30 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, Lys,
Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
alpha, gamma-diaminobutryic acid, or homoglutamic acid;
Xaa at position 31 is Trp, Phe, Tyr, Glu, Asp, or Lys;
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Xaa at position 32 is Leu, Gly, Ala, Ser, Thr, Ile, Val, Glu, Asp, or Lys;
Xaa at position 33 is Val, Gly, Ala, Ser, Thr, Leu, Ile, Glu, Asp, or Lys;
Xaa at position 34 is Asn, Lys, Arg, Glu, Asp, or His;
Xaa at position 35 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 36 is Gly, Arg, Lys, Glu, Asp, or His;
Xaa at position 37 is Pro, Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, or
Lys;
Xaa at position 38 is Gly, Ser, Lys, Cys, or is omitted;
Xaa at position 39 is Gly, Ala, Ser, Thr, Ile, Val, Leu, Phe, Pro, Cys or is
omitted;
Xaa at position 40 is Gly, Cys, or is omitted;
Xaa at position 41 is Gly or is omitted;
wherein two amino acids selected from Xaa18, Xaa19, Xaa20, Xaa21, Xaa22,
Xaa23, Xaa24,
Xaa25, Xaa26, Xaa27, Xaa28, Xaa29, and Xaa30 are joined to form a ring and the
two amino
acids forming the ring are separated by 0, 1, 2, 3, 4 or 5 amino acids, and
wherein the
compound has a GLP-1 activity. In a particular aspect, the GLP-1 analog has
the amino
acid sequence of any of SEQ ID NO: 30 to 246 with no more than 5 conservative
amino
acid substitutions. In another particular aspect, the GLP-1 analog has the
amino acid
sequence of any of SEQ ID NO: 30 to 246.
In still another aspect, a GLP-1 compound as provided herein comprises a GLP-1
analog comprising the amino acid sequence shown in formula VI:
Xaa4-Xaa5-Xaa6-His7-Ala8-Glu9-Gly 0-Thr 1-Phe 2-Thr 3-Ser 4-Asp 5-Val 6-Seri 7-
Seriii-TYri9-LeU2O-GIU21-Gly22-Gin23-Ala24-Ala25-Lys26-Glu27-Phe28-Ile29-Ala30-
Trp3i-Leu32-Va133-Lys34-Gly35-Arg36-C(0)-R (Formula VI, SEQ ID NO: 276)
wherein,
R1 is OR2 or NR2R3;
R2 and R3 are independently hydrogen or (CI-C8)alkyl;
Xaa at position 4 is: Met or omitted;
Xaa at position 5 is: Met, His, or omitted;
Xaa at position 6 is: Met, Ala, Gly, Pro, Ser, Thr, Val, Gin, Arg, Lys, His,
Tyr,
Ile, Asp, Leu, Asn, Glu, Trp, or Phe;
provided that when the amino acid at position 5 is omitted, then the amino
acid at
position 4 is also omitted, wherein the compound has a GLP-1 activity.
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Also provided are pharmaceutical compositions comprising a pharmaceutically
acceptable carrier and an effective amount of a GLP-1 compound as described
herein.
In addition, methods are provided for treating a subject with a metabolic
disorder,
comprising administering to the subject an effective amount of a GLP-1
compound as
provided herein or a pharmaceutical composition comprising a GLP-1 compound as
provided herein, wherein the metabolic disorder is selected from the group of
diabetes,
obesity and metabolic syndrome.
In addition, methods are provided for enhancing insulin expression in a
subject
and methods for promoting insulin secretion in a subject, comprising
administering to the
subject an effective amount of a GLP-1 compound as provided herein or a
pharmaceutical composition comprising a GLP-1 compound as provided herein.
In certain aspects, a GLP-1 compound as provided herein can be covalently
modified with a water-soluble polymer, such as polyethylene glycol,
monomethoxy-
polyethylene glycol, dextran, cellulose, poly-(N-vinyl pyrrolidone)
polyethylene glycol,
propylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers,
polyoxyethylated polyols, or polyvinyl alcohol.
Specific embodiments will become evident from the following more detailed
description of certain embodiments and the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic representation of examples of the positions within GLP-
1
separated by 3 amino acids that can be joined to form a cyclic lactam.
Figures 2A through 21 depict chemical structures of exemplary GLP-1 analogs in
which the side chains of a glutamic acid/ lysine amino acid pair are joined to
form a ring.
GLP-1 residues are shown in single letter code, whereas atoms for expanded
amino acids
are shown using normal chemical abbreviations for the elements. The number of
ethylene glycol repeating units -[CH2CH2-01n- can vary depending on the size
of
polyeyhleneglycol desired (e.g, 5kDa to 60kDa).
Figure 3 illustrates an exemplary approach for synthesizing an analog that
includes a Gly substitution at position 8, an Aib substitution at position 22
and the
addition of a cysteine and an alanine at the C-terminus (i.e., SEQ ID NO:18).
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Figure 4 provides an exemplarly synthetic scheme for preparing a cyclic GLP-1
compound in which the side chains of a glutamic acid and lysine residue are
joined to
form a cyclic lactam.
Figure 5 provides an exemplarly synthetic scheme for preparing a cyclic GLP-1
compound in which the side chains of glutamic acid and ornithine are joined to
form a
cyclic lactam.
Figure 6 provides an exemplarly synthetic scheme for preparing a cyclic GLP-1
compound in which the side chains of an aspartic acid and lysine residue are
joined to
form a cyclic lactam.
Figure 7 provides an exemplarly synthetic scheme for preparing a cyclic GLP-1
compound in which the side chains of aspartic acid and ornithine are joined to
form a
cyclic lactam.
Figure 8 provides an exemplarly synthetic scheme for preparing a cyclic GLP-1
compound in which the side chains of a lysine and glutamic acid residue are
joined to
form a cyclic lactam.
Figure 9 provides an exemplarly synthetic scheme for preparing a cyclic GLP-1
compound in which the side chains of homoglutamic acid and lysine are joined
to form a
cyclic lactam.
Figure 10 provides an exemplarly synthetic scheme for preparing a cyclic GLP-1
compound in which the side chains of 4-carboxy-phenylalanine and lysine are
joined to
form a cyclic lactam.
Figure 11 provides an exemplarly synthetic scheme for preparing a cyclic GLP-1
compound in which the side chains of beta-Homoglutamic acid and lysine are
joined to
form a cyclic lactam.
Figure 12 shows a graph depicting blood glucose levels in mice treated with
cgGLP-17, cgGLP-18, cgGLP-19, or cgGLP-09A.
Figure 13 shows a graph depicting blood glucose levels in mice treated with
cgGLP-19, cgGLP-20, cgGLP-23, or cgGLP-24.
Figure 14 shows a graph depicting blood glucose levels in mice treated with
mgGLP-20, cgGLP-26, mgGLP-22, or mgGLP-24.
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Figure 15 shows a graph depicting blood glucose levels in mice treated with
mgGLP32.
Figure 16 shows a graph depicting blood glucose levels during a set of OTT
(glucose tolerance test) experiments conducted with mice treated with mgGLP-
20,
mgGLP-22 or mgGLP-24.
Figure 17 shows a graph depicting blood glucose levels in mice treated with
mgGLP-33.
Figure 18 shows a graph depicting blood glucose levels in mice treated with
mgGLP-32.
Figure 19 shows a graph depicting blood glucose levels in mice treated with
mgGLP-20.
Figure 20 shows a graph depicting the effect of PEG size and shape on certain
cgGLP-3, cgGLP-1, cgGLP-7 and cgGLP-2.
Figure 21 shows a graph depicting the effect of PEG size and shape on cgGLP-6,
cgGLP-4, cgGLP-8, cgGLP-5.
Figure 22 shows a graph depicting the effect of PEG size and shape on cgGLP-
25,
cgGLP-26, cgGLP-24, cgGLP-28, cgGLP-29.
Figure 23 shows a graph depicting the effect of PEG size and shape in vivo on
db/db mice treated with a GLP-1 compound having the amino acid sequence set
forth in
SEQ ID NO:22 and either a cgGLP-25, cgGLP-26, cgGLP-27, cgGLP-29.
DETAILED DESCRIPTION
I. Definitions
As used in this specification and the appended claims, the singular forms "a,"
"an"
and "the" include plural references unless the content clearly dictates
otherwise.
Unless defined otherwise, all technical and scientific terms used herein have
the
meaning commonly understood by a person skilled in the art to which this
invention
belongs. The following references provide one of skill with a general
definition of many
of the terms used in this invention: Singleton et al., DICTIONARY OF
MICROBIOLOGY AND MOLECULAR BIOLOGY (2d ed. 1994); THE CAMBRIDGE
DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1988); THE
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GLOSSARY OF GENETICS, 5TH ED., R. Rieger et al. (eds.), Springer Verlag
(1991);
and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY (1991).
As used herein, the following terms have the meanings ascribed to them unless
specified otherwise.
"Insulinotropic activity" refers to the ability to increase insulin synthesis,
release
or secretion in a glucose-dependent manner. The insulinotropic effect can
result from
any of a number of different mechanisms, including, but not limited to, an
increase in the
number of insulin positive cells and/or due to an increase in the amount of
insulin
synthesized or released from existing insulin positive cells in a given time
period.
Insulinotropic activity can be assayed using methods known in the art, such as
in vivo and
in vitro experiments that measure GLP-1 receptor binding activity or receptor
activiation
(for example, assays using pancreatic islet cells or insulinoma cells as
described in EP
619,322 and US Patent No. 5,120,712). In humans, insulinotropic activity can
be
measured by examining insulin levels or C-peptide levels.
The terms "nucleic acid," "polynucleotide," and "oligonucleotide" are used
herein
to include a polymeric form of nucleotides of any length, including, but not
limited to,
ribonucleotides or deoxyribonucleotides.
"Polypeptide" and "protein" are used interchangeably herein and include a
molecular chain of amino acids linked through peptide bonds. The terms do not
refer to a
specific length of the product. Thus, "peptides," and "oligopeptides," are
included within
the definition of polypeptide. The terms include post-translational
modifications of the
polypeptide, for example, glycosylations, acetylations, phosphorylations and
the like. In
addition, protein fragments, analogs, mutated or variant proteins, fusion
proteins and the
like are included within the meaning of polypeptide. The terms also include
molecules in
which one or more amino acid analogs or unnatural amino acids are included.
The terms "identical" or percent "identity," in the context of two or more
nucleic
acids or polypeptides, refer to two or more sequences or subsequences that are
the same
or have a specified percentage of nucleotides or amino acid residues that are
the same,
when compared and aligned for maximum correspondence, as measured using a
sequence
comparison algorithm such as those described below for example, or by visual
inspection.
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The phrase ''substantially identical" as used herein refers to two or more
sequences or subsequences that have at least 75%, preferably at least 85%,
more
preferably at least 90%, 95%, 96%, 97%, 98%, 99% or higher nucleotide or amino
acid
residue identity, when compared and aligned for maximum correspondence, as
measured
using a sequence comparison algorithm such as those described below for
example, or by
visual inspection. Most preferably the sequences are substantially identical
over the full
length of the sequences being compared, such as the coding region of a
nucleotide for
example.
For sequence comparison, typically one sequence acts as a reference sequence,
to
which test sequences are compared. When using a sequence comparison algorithm,
test
and reference sequences are input into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters are
designated. The
sequence comparison algorithm then calculates the percent sequence identity
for the test
sequence(s) relative to the reference sequence, based on the designated
program
parameters.
Optimal alignment of sequences for comparison can be conducted, e.g., by the
local homology algorithm of Smith & Waterman, 1981, Adv. App!. Math. 2:482, by
the
homology alignment algorithm of Needleman & Wunsch, 1970õ/ Mol. Biol. 48:443,
by
the search for similarity method of Pearson & Lipman, 1988, Proc. Nat'l. Acad.
Sci. USA
85:2444, by computerized implementations of these algorithms (GAP, BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer
Group, 575 Science Dr., Madison, Wis.), or by visual inspection [see
generally, Current
Protocols in Molecular Biology, (Ausubel, F. M. et al., eds.) John Wiley &
Sons, Inc.,
New York (1987-1999, including supplements such as supplement 46 (April
1999)]. Use
of these programs to conduct sequence comparisons are typically conducted
using the
default parameters specific for each program.
Another example of algorithm that is suitable for determining percent sequence
identity and sequence similarity is the BLAST algorithm, which is described in
Altschul
et al., 1990, J Mal, Biol. 215:403-410. Software for performing BLAST analyses
is
publicly available through the National Center for Biotechnology Information.
This
algorithm involves first identifying high scoring sequence pairs (HSPs) by
identifying
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short words of length W in the query sequence, which either match or satisfy
some
positive-valued threshold score T when aligned with a word of the same length
in a
database sequence. T is referred to as the neighborhood word score threshold
(Altschul
et al, supra.). These initial neighborhood word hits act as seeds for
initiating searches to
find longer HSPs containing them. The word hits are then extended in both
directions
along each sequence for as far as the cumulative alignment score can be
increased.
Cumulative scores are calculated using, for nucleotide sequences, the
parameters M
(reward score for a pair of matching residues; always >0) and N (penalty score
for
mismatching residues; always <0). For amino acid sequences, a scoring matrix
is used to
calculate the cumulative score. Extension of the word hits in each direction
are halted
when: the cumulative alignment score falls off by the quantity X from its
maximum
achieved value; the cumulative score goes to zero or below, due to the
accumulation of
one or more negative-scoring residue alignments; or the end of either sequence
is
reached. For identifying whether a nucleic acid or polypeptide is within the
scope of the
invention, the default parameters of the BLAST programs are suitable. The
BLASTN
program (for nucleotide sequences) uses as defaults a word length (W) of 11,
an
expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino
acid
sequences, the BLASTP program uses as defaults a word length (W) of 3, an
expectation
(E) of 10, and the BLOSUM62 scoring matrix. The TBLATN program (using protein
sequence for nucleotide sequence) uses as defaults a word length (W) of 3, an
expectation
(E) of 10, and a BLOSUM 62 scoring matrix. (see Henikoff & Henikoff, 1989,
Proc.
Natl. Acad. Sci. USA 89:10915).
In addition to calculating percent sequence identity, the BLAST algorithm also
performs a statistical analysis of the similarity between two sequences (see,
e.g., Karlin &
Altschul, 1993, Proc. Nat'l, Acad. Sci. USA 90:5873-5787). One measure of
similarity
provided by the BLAST algorithm is the smallest sum probability (P(N)), which
provides
an indication of the probability by which a match between two nucleotide or
amino acid
sequences would occur by chance. For example, a nucleic acid is considered
similar to a
reference sequence if the smallest sum probability in a comparison of the test
nucleic acid
to the reference nucleic acid is less than about 0.1, more preferably less
than about 0.01,
and most preferably less than about 0.001.
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A polypeptide is typically substantially identical to a second polypeptide,
for
example, where the two polypeptides differ only by conservative substitutions.
A
"conservative substitution" when describing a protein, refers to a change in
the amino
acid composition of the protein that does not substantially alter the
protein's activity.
Thus, "conservatively modified variations" of a particular amino acid sequence
refers to
amino acid substitutions of those amino acids that are not critical for
protein activity or
substitution of amino acids with other amino acids having similar properties
(e.g., acidic,
basic, positively or negatively charged, polar or non-polar, etc.) such that
the
substitutions of even critical amino acids do not substantially alter
activity. Conservative
substitution tables providing functionally similar amino acids are described
herein and
are well-known in the art (see, e.g., Creighton, 1984, Proteins, W.H. Freeman
and
Company). In addition, individual substitutions, deletions or additions that
alter, add or
delete a single amino acid or a small percentage of amino acids in an encoded
sequence
without substantially altering the protein's activity are also "conservatively
modified
variations."
As used herein, the twenty conventional amino acids and their single letter
and
three letter abbreviations follow conventional usage.
See IMMUNOLOGY--A
SYNTHESIS, 2nd Edition, (E. S. Golub and D. R. Gren, Eds.), Sinauer
Associates:
Sunderland, MA, 1991, incorporated herein by reference for any purpose.
Stereoisomers
(e.g., D-amino acids) of the twenty conventional amino acids; unnatural amino
acids such
as a-, a-disubstituted amino acids, N-alkyl amino acids, lactic acid, and
other
unconventional amino acids may also be suitable components for polypeptides
provided
herein.
Examples of unconventional amino acids include: 4-hydroxyproline, 7-
carboxyglutamate, E-N,N,N-trimethyllysine, E-N-acetyllysine, 0-phosphoserine,
N-
acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, a-N-
methylarginine, and other similar amino acids and imino acids (e.g., 4-
hydroxyproline).
Certain amino acid analogs that are referrenced herein are abbreviated as
follows:
bAla is beta-aminopropionic acid;
J is Aad (2-aminoadipic acid; also called homoglutamic acid);
Z is Aib (2-aminoisobutyric acid);
0 is ornithine;
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Cpa is 4-carboxy-phenylalanine; and
B is beta glutamic acid.
When amino acids abbreviations are separated by a forward slash (i.e., a "/"),
this means
that any one of the amino acids separated by the forward slash can occur at
the indicated
position. For instance, K/O/C means that any one of lysine, ornithine or
cysteine can
occur at the indicated position.
The term alpha-alpha disubstituted amino acids as used herein has its normal
meaning in the art and includes, for example, alpha-methyl-leucine, alpha-
methyl-
phenylalanine, alpha-methyl-tryptophan, 4-amino-l-piperdine, 2-
amino-2,2-
diphenylacetic acid.
In the polypeptide notation used herein, the left-hand direction is the amino
terminal direction and the right-hand direction is the carboxyl-terminal
direction, in
accordance with standard usage and convention. The term "downstream" when used
in
reference to a GLP-1 compound means positions that are located toward the
carboxyl end
of the polypeptide relative to the position being referenced, i.e., to the
right of the
position being referenced. The term "upstream" when used in reference to a GLP-
1
compound means positions that are located toward the amino terminal end of the
polypeptide relative to the position being referenced, i.e., to the left of
the position being
referenced. The recommended IllPAC-IIJB Nomenclature and Symbolism for Amino
Acids and Peptides have been published in J. Biochem., 1984, 219, 345-373;
Eur. J.
Biochem., 1984, 138, 9-37; 1985, 152, 1; 1993, 213, 2; Internat. J. Pept.
Prot. Res., 1984,
24, following p 84; J. Biol. Chem., 1985, 260,14-42; Pure Appl. Chem., 1984,
56, 595-
624; Amino Acids and Peptides, 1985, 16, 387-410; Biochemical Nomenclature and
Related Documents, 2nd edition, Portland Press, 1992, pages 39-69.
Naturally occurring residues may be divided into classes based on common side
chain properties:
1) hydrophobic: norleucine (Nor), Met, Ala, Val, Leu, Ile;
2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
3) acidic: Asp, Glu;
4) basic: His, Lys, Arg;
5) residues that influence chain orientation: Gly, Pro; and
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6) aromatic: Trp, Tyr, Phe.
Conservative amino acid substitutions may involve exchange of a member of one
of these classes with another member of the same class. Conservative amino
acid
substitutions may encompass non-naturally occurring amino acid residues, which
are
typically incorporated by chemical peptide synthesis rather than by synthesis
in
biological systems. These include peptidomimetics and other reversed or
inverted forms
of amino acid moieties.
Non-conservative substitutions may involve the exchange of a member of one of
these classes for a member from another class. Such substituted residues may
be
introduced into regions of the human antibody that are homologous with non-
human
antibodies, or into the non-homologous regions of the molecule.
In making such changes, according to certain embodiments, the hydropathic
index
of amino acids may be considered. Each amino acid has been assigned a
hydropathic
index on the basis of its hydrophobicity and charge characteristics. They are:
isoleucine
(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine
(+2.5);
methionine (+1.9); alanine (+1.8); glycine (-0.4); tlueonine (-0.7); serine (-
0.8);
tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2);
glutamate (-3.5);
glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and
arginine (-4.5).
The importance of the hydropathic amino acid index in conferring interactive
biological function on a protein is understood in the art (see, for example,
Kyte et al.,
1982, J. Mol. Biol. 157:105-131). It is known that certain amino acids may be
substituted
for other amino acids having a similar hydropathic index or score and still
retain a similar
biological activity. In making changes based upon the hydropathic index, in
certain
embodiments, the substitution of amino acids whose hydropathic indices are
within +2 is
included. In certain embodiments, those that are within 1 are included, and
in certain
embodiments, those within +0.5 are included.
It is also understood in the art that the substitution of like amino acids can
be
made effectively on the basis of hydrophilicity, particularly where the
biologically
functional protein or peptide thereby created is intended for use in
immunological
embodiments, as disclosed herein. In certain embodiments, the greatest local
average
hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent
amino acids,
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correlates with its irnmunogenicity and antigenicity, i.e., with a biological
property of the
protein.
The following hydrophilicity values have been assigned to these amino acid
residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 1); glutamate
(+3.0 1); serine
(+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4);
proline (-0.5
1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3);
valine (-1.5); leucine
(-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and
tryptophan (-3.4). In
making changes based upon similar hydrophilicity values, in certain
embodiments, the
substitution of amino acids whose hydrophilicity values are within 2 is
included, in
certain embodiments, those that are within 1 are included, and in certain
embodiments,
those within 0.5 are included. One may also identify epitopes from primary
amino acid
sequences on the basis of hydrophilicity. These regions are also referred to
as "epitopic
core regions."
Exemplary amino acid substitutions are set forth in Table 1.
Table 1
Amino Acid Substitutions
Preferred
Original Residues Exemplary Substitutions
Substitutions
Ala Val, Leu, Ile Val
Arg Lys, Gin, Asn Lys
Asn Gin Gin
Asp Glu Glu
Cys Ser, Ala Ser
Gin Asn Asn
Glu Asp Asp
Gly Pro, Ala Ala
His Asn, Gin, Lys, Arg Arg
Leu, Val, Met, Ala,
Ile Leu
Phe, Norleucine
Norieucine, Ile, Val,
Leu Ile
Met, Ala, Phe
Arg, 1,4 Diamino-
Lys Arg
butyric Acid, Gin, Asn
Met Leu, Phe, Ile Leu
Phe Leu, Val, Ile, Ala, Tyr Leu
Pro Ala Gly
Ser Thr, Ala, Cys Thr
Thr Ser Ser
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Preferred
Original Residues Exemplary Substitutions
Substitutions
Trp Tyr, Phe Tyr
Tyr Trp, Phe, Thr, Ser Phe
Ile, Met, Leu, Phe, Ala,
Val Leu
Norleucine
Herein, the use of the protecting groups, linkers, and solid phase supports,
as well
as specific protection and deprotection reaction conditions, linker cleavage
conditions,
use of scavengers, and other aspects of solid phase peptide synthesis are well
known and
are also described in "Protecting Groups in Organic Synthesis," 3rd Edition,
T. W.
Greene and P. G. M. Wuts, Eds., John Wiley & Sons, Inc., 1999; NovaBiochem
Catalog,
2000; "Synthetic Peptides, A User's Guide," G. A. Grant, Ed., W.H. Freeman &
Company, New York, N.Y., 1992; "Advanced Chemtech Handbook of Combinatorial &
Solid Phase Organic Chemistry," W. D. Bennet, J. W. Christensen, L. K.
Hamaker, M. L.
Peterson, M. R. Rhodes, and H. H. Saneii, Eds., Advanced Chemtech, 1998;
"Principles
of Peptide Synthesis, 2nd ed.," M. Bodanszky, Ed., Springer-Verlag, 1993; "The
Practice
of Peptide Synthesis, 2nd ed.," M. Bodanszky and A. Bodanszky, Eds., Springer-
Verlag,
1994; "Protecting Groups," P. J. Kocienski, Ed., Georg Thieme Verlag,
Stuttgart,
Germany, 1994; "Fmoc Solid Phase Peptide Synthesis, A Practical Approach," W.
C.
Chan and P. D. White, Eds., Oxford Press, 2000, G. B. Fields et al., Synthetic
Peptides: A
User's Guide, 1990, 77-183, and elsewhere.
A skilled artisan will be able to determine suitable variants of the
polypeptide as
set forth herein using well-known techniques. In certain embodiments, one
skilled in the
art may identify suitable areas of the molecule that may be changed without
destroying
activity by targeting regions not believed to be important for activity. In
other
embodiments, the skilled artisan can identify residues and portions of the
molecules that
are conserved among similar polypeptides. In further embodiments, even areas
that may
be important for biological activity or for structure may be subject to
conservative amino
acid substitutions without destroying the biological activity or without
adversely affecting
the polypeptide structure.
Additionally, one skilled in the art can review structure-function studies
identifying residues in similar polypeptides that are important for activity
or structure. In
24
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view of such a comparison, the skilled artisan can predict the importance of
amino acid
residues in a protein that correspond to amino acid residues important for
activity or
structure in similar proteins. One skilled in the art may opt for chemically
similar amino
acid substitutions for such predicted important amino acid residues.
One skilled in the art can also analyze the three-dimensional structure and
amino
acid sequence in relation to that structure in similar polypeptides. In view
of such
information, one skilled in the art may predict the alignment of amino acid
residues of an
antibody with respect to its three dimensional structure. In certain
embodiments, one
skilled in the art may choose to not make radical changes to amino acid
residues
predicted to be on the surface of the protein, since such residues may be
involved in
important interactions with other molecules. Moreover, one skilled in the art
may
generate test variants containing a single amino acid substitution at each
desired amino
acid residue. The variants can then be screened using activity assays known to
those
skilled in the art. Such variants could be used to gather information about
suitable
variants. For example, if one discovered that a change to a particular amino
acid residue
resulted in destroyed, undesirably reduced, or unsuitable activity, variants
with such a
change can be avoided. In other words, based on information gathered from such
routine
experiments, one skilled in the art can readily determine the amino acids
where further
substitutions should be avoided either alone or in combination with other
mutations.
A number of scientific publications have been devoted to the prediction of
secondary structure. See, for example, Moult, 1996, Curr, Op. in Biotech.
7:422-427;
Chou et al., 1974, Biochemistry 13:222-245; Chou et al., 1974, Biochemistry
113:211-
222; Chou et al., 1978, Adv. Enzymol. Relat. Areas Mol. Biol, 47:45-148; Chou
et al.,
1979, Ann. Rev. Biochem. 47:251-276; and Chou et al., 1979, Biophys, J. 26:367-
384.
Moreover, computer programs are currently available to assist with predicting
secondary
structure. One method of predicting secondary structure is based upon homology
modeling. For example, two polypeptides or proteins that have a sequence
identity of
greater than 30%, or similarity greater than 40% often have similar structural
topologies.
The recent growth of the protein structural database (PDB) has provided
enhanced
predictability of secondary structure, including the potential number of folds
within a
polypeptide's or protein's structure. See Holm et al., 1999, Nucl. Acid. Res.
27:244-247.
CA 02648936 2011-06-02
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It has been suggested (Brenner et al., 1997, Curr. Op. Struct. Biol. 7:369-
376) that there
are a limited number of folds in a given polypeptide or protein and that once
a critical
number of structures have been resolved, structural prediction will become
dramatically
more accurate.
Additional methods of predicting secondary structure include "threading"
(Jones,
1997, Cum Opin. Struct. Biol. 7:377-87; Sippl et al., 1996, Structure 4:15-
19), "profile
analysis" (Bowie et al., 1991, Science 253:164-170; Gribskov et al., 1990,
Meth. Enzym.
183:146-159; Gribskov et al., 1987, Proc. Nat. Acad. Sc!. 84:4355-4358), and
"evolutionary linkage" (See Holm, 1999, supra; and Brenner, 1997, supra).
According to certain embodiments, amino acid substitutions are those that: (1)
reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation,
(3) alter
binding affinity for forming protein complexes, (4) alter binding affinities,
and/or (5)
confer or modify other physicochemical or functional properties on such
polypeptides.
According to certain embodiments, single or multiple amino acid substitutions
(in certain
embodiments, conservative amino acid substitutions) may be made in the
naturally
occurring sequence (in certain embodiments, in the portion of the polypeptide
outside the
domain(s) forming intermolecular contacts). In certain embodiments, a
conservative
amino acid substitution typically does not substantially change the structural
characteristics of the parent sequence (e.g., a replacement amino acid should
not tend to
break a helix that occurs in the parent sequence, or disrupt other types of
secondary
structure that characterizes the parent sequence). Examples of art-recognized
polypeptide
secondary and tertiary structures are described in PROTEINS, STRUCTURES AND
MOLECULAR PRINCIPLES, (Creighton, Ed.), 1984, W. H. Freeman and Company,
New York; INTRODUCTION TO PROTEIN STRUCTURE (C. Branden and J. Tooze,
eds.), 1991, Garland Publishing, New York, N.Y.; and Thornton et al., 1991,
Nature
354:105,
Peptide analogs are commonly used in the pharmaceutical industry as non-
peptide
drugs with properties analogous to those of the template peptide. These types
of non-
peptide compound are termed "peptide mimetics" or "peptidomimetics". See
Fauchere,
1986, Adv. Drug Res, 15:29; Veber & Freidinger, 1985, TINS p.392; and Evans et
al,.
1987, J. Med Chem. 30:1229,
26
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Such compounds are often developed with the aid of computerized molecular
modeling. Peptide mimetics that are structurally similar to therapeutically
useful peptides
may be used to produce a similar therapeutic or prophylactic effect.
Generally,
peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a
polypeptide
that has a biochemical property or pharmacological activity), such as human
antibody,
but have one or more peptide linkages optionally replaced by a linkage
selected from; -
CH2-NH-, -CH2-S-, -CH2-CH2-, -CH=CH-(cis and trans), -COCH2-, -CH(OH)CH2-, and
-CH2S0-, by methods well known in the art. Systematic substitution of one or
more
amino acids of a consensus sequence with a D-amino acid of the same type
(e.g., D-lysine
in place of L-lysine) may be used in certain embodiments to generate more
stable
peptides. In addition, constrained peptides comprising a consensus sequence or
a
substantially identical consensus sequence variation may be generated by
methods known
in the art (Rizo & Gierasch, 1992, Ann. Rev. Biochem. 61:387); for example, by
adding
internal cysteine residues capable of
forming intramolecular disulfide bridges which cyclize the peptide.
II. Overview
A variety of GLP-1 compounds are provided herein that comprise a GLP-1
analog, and which retain at least one activity of GLP-1. The GLP-1 analogs
that are
disclosed include one or more of the following characteritics; 1) amino acid
substitutions
at particular locations of GLP-1, 2) added amino acids at the N-terminus
and/or the C-
terminus of GLP-1, 3) absence of amino acids at the N-terminus and/or the C-
terminus of
GLP-1, and/or 4) presence of a ring formed by joining the side chains of
specific amino
acids with the polypeptide.
As described in greater detail below, the GLP-1 compounds that are provided
can
be administered therapeutically or prophylactically to treat a variety of
diseases.
Examples of diseases that can be treated with the compounds include, but are
not limited
to, diabetes, impaired glucose tolerance, insulin resistance, hyperglycemia,
metabolic
syndrome, various lipid disorders, obesity, coronary diseases, bone disorders,
and
irritable bowel syndrome.
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III. GLP-1 Compounds
A. Structure
As noted above, the term "GLP-1" refers to GLP-1 (7-37)-OH and GLP-1 (7-36)-
NH2. The numbering of the amino acids of GLP-1 as used herein is based on GLP-
1 (1-
37) formed from cleavage of proglucagon.
Native GLP-1 (7-37)-OH has the following amino acid sequence:
7His-8Ala-9G1u- I Gly- " Thr- I 2Phe-13Thr-14Ser- I 5 Asp-1 6Val- I 7Ser-1 8S
er- I 9Tyr-20Leu-2 Glu-
22Gly_23Giu_24Aia_25ma_26Lys_27 -u_
28Phe-29Ile-3 Ala-31Trp-32Leu-33Val-34Lys-35Gly-
36Arg-37Gly (SEQ ID NO: 1).
As indicated in this formula, the amino terminal His residue is customarily
referred to as amino acid residue 7 to reflect processing from GLP-1 (1-37);
the carboxyl
terminal Gly in turn is conventionally referred to as amino acid residue 37.
As described
in the Background section, the carboxyl terminus of GLP-1 (7-37)-OH can be
cleaved to
produce GLP-1 (7-36)-NH2. The other amino acids located between these two
termini
are numbered consecutively as shown. Thus, for example, the amino acid at
position 8 is
Ala and the amino acid at position 26 is Lys. Likewise, when reference is made
herein to
making a substitution at a specified position, the same numbering system
applies. Hence,
for example, a substitution of Ala at position 22 means that the Gly at
position 22 has
been substituted with Ala. If amino acids are added at the amino terminus of
GLP-1 (7-
36), the positions are consecutively numbered in decreasing order, such that
the amino
acid immediately upstream of position 7 is amino acid 6, and the next upstream
amino
acid is at position 5 and so on. If amino acids are added at the carboxyl
terminus of GLP-
1 (7-36), the positions are consecutively numbered in increasing order, such
that the
amino acid immediately downstream of position 36 is amino acid 37, and the
next
downstream amino acid is at position 38 and so on. As discussed above, both
GLP-1 (7-
37)-OH, also referred to as GLP-1 (7-37), and GLP-1 (7-36)-NH2 have the same
activities. For convenience, the terms "GLP-1" and "native GLP-1" are used to
refer to
both of these biologically active forms.
A "GLP-1 compound" as used herein refers to a molecule that comprises a GLP-1
analog and may include one or more additional components (e.g., a component
that
extends the half-life of the compound in vivo).
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The phrase "GLP-1 activity" or grammatical equivalents thereof refers broadly
to
any activity associated with GLP-1. Examples of such activities include, but
are not
limited to, insulinotropic activity, inhibition of gastric motility,
inhibition of gastric
secretion, promotion of 13-cell proliferation and replication, increase in 13-
cell mass,
increase in satiety and decrease in food intake when GLP-1 is administered to
a subject.
As used herein, the term "GLP-1 analog" refers to a polypeptide with one or
more
alterations in the amino acid sequence of native GLP-1 (7-37)-OH or GLP-1 (7-
36)-NH2
but that retains at least one activity of native GLP-1. The GLP-1 analogs as
provided
herein as described herein include, for example, specific amino acid
substitutions at
particular residues of GLP-1. Some of the analogs also include added amino
acid
residues at the C-terminus, whereas others are shortened at the C-terminus.
The GLP-1
analogs as provided herein markedly decrease blood glucose levels in various
in vivo
models and have extended half-lives relative to native GLP-1. As described
herein, the
GLP-1 analogs can be pegylated with one or more molecules of polyethylene
glycol
(PEG) to increase the in vivo half-life of the analog. Alternatively, GLP-1
analogs can be
joined to another polypeptide to form a fusion protein, as described herein.
In the
formulas listed herein, R1 can be a carboxyl group, an amine, an ester, or a
substituted
amine. Thus, the GLP-1 analogs as provided herein can have a carboxyl or an
amide
group at its C-terminal end.
In certain embodiments, a GLP-1 analog as provided herein has one or more of
the following characteristics:
1) One or more amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10) relative to GLP-1;
2) The addition of one or more additional amino acid residues at the C-
terminus or N-terminus of GLP-1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino
acids);
3) Truncation of one or more amino acids at the N- or C-terminus
(typically by 1, 2, 3, 4 or 5 amino acids); and/or
4) A cyclic ring structure formed between side-chains on certain amino
acids within the analog.
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Certain GLP-1 analogs that are provided, for instance, include a specific
amino
acid substitution at one or more of positions 8, 22, 23, 26, 33, 34, 35, 36,
and/or 37,
including, but not limited to, the following substitutions:
8Ala: substitution with Gly, 2-aminoisobutryic acid (Aib) or beta-
aminopropionic
acid (bAla);
: substitution with 2-aminoisobutyric acid (Aib), 1-amino-
cylcopentanecarboxylic acid, an alpha-alpha-disubstituted amino acid, or 2-
aminoadipic
acid;
23G1n: substitution with Cys;
26Lys: substitution with ornithine or homolysine;
33Val: substitution with Lys;
34Lys: substitution with Asn;
36Arg: substitution with Gly;
37Gly: substitution with Pro.
Some of the GLP-1 analogs include specific C-terminal extensions, including,
for
instance, the addition of Cys, Cys-Ala, Cys-Gly, Cys-Ser-Gly, Cys-Ser-Gly-Gly
(SEQ ID
NO: 278), or Gly-Cys (each of the foregoing being listed in the amino to
carboxyl
direction) to the amino acid at position 37.
Other GLP-1 analogs include specific N-terminal additions such as the addition
of
Met, Ala, Gly, Pro, Ser, Thr, Val, Gln, Arg, Lys, His, Tyr, Ile, Asp, Leu,
Asn, Glu, Trp or
Phe at the N-terminus of GLP-1. Some analogs are extended at the N-terminus by
the
addition of a MQ, MR, MK, MH, MY, MI, MD, ML, MN, ME, MW, MF or MM
dipeptide to the amino terminus. Still other analogs have a MHH tripeptide
added to the
amino terminus.
The GLP-1 analogs can for ease of discussion be classified into certain
families of
molecules that share certain structural features. These can be most readily
described by
the general Formulas provided as follows.
One family, for instance, comprises or consists of the amino acid sequence
shown
in formula I:
Xaa7-Gly-Xaa9-Xaaio-Xaai 1-Xaa12-Xaa13-Xaa14-Xaai 5-Xaa1 6-Xaa17-Xaa1 8_Xaa19-
Xaa20-Xaa21-Xaa22-Cys-Xaa24-Xaa25-Xaa26-Xaa27_Xaa28-Xaa29-Xaa30_Xaa31-
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Xaa32-Xaa33_Xaa34_Gly_Xaa36Aaar-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-C(0)-Ri
(Formula I, SEQ ID NO: 5)
wherein,
R1 is OR2 or NR2R3;
R2 and R3 are independently hydrogen or (CI -C8)alkyl;
Xaa at position 7 is: L-histidine, D-histidine, desamino-histidine, 2-amino-
histidine, 3-hydroxy-histidine, homohistidine, a-fluoromethyl-histidine or a-
methyl-histidine;
Xaa at position 9 is Glu, Asp, or Lys;
Xaa at position 10 is Gly or His;
Xaa at position 11 is Thr, Ala, Gly, Ser, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 12 is: His, Trp, Phe, or Tyr;
Xaa at position 13 is Thr or Gly;
Xaa at position 14 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 15 is Asp or Glu;
Xaa at position 16 is Val, Ala, Gly, Ser, Thr, Leu, Ile, Tyr, Glu, Asp, Trp,
or Lys;
Xaa at position 17 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 18 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, Trp, Tyr,
Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-
Homoglutamic acid, or homoglutamic acid;
Xaa at position 19 is Tyr, Phe, Trp, Glu, Asp, Gin, Lys, Homolysine,
Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or
homoglutamic acid;
Xaa at position 20 is Leu, Ala, Gly, Ser, Thr, Ile, Val, Glu, Asp, Met, Trp,
Tyr,
Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, or
homoglutamic acid;
Xaa at position 21 is Glu, Asp, Lys, Homolysine, Ornithine, 4-carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic
acid;
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Xaa at position 22 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, Lys,
Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
or homoglutamic acid;
Xaa at position 24 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Arg, Glu, Asp, Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-
Homoglutamic acid, or homoglutamic acid;
Xaa at position 25 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, Lys,
Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
or homoglutamic acid;
Xaa at position 26 is Lys, Homolysine, Arg, Gln, Glu, Asp, His, Ornithine, 4-
carboxy-phenylalanine, beta-glutamic acid, or homoglutamic acid;
Xaa at position 27 is Leu, Glu, Asp, Lys, Homolysine, Ornithine, 4-carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic
acid;
Xaa at position 28 is Phe, Trp, Asp, Glu, Lys, Homolysine, Ornithine, 4-
carboxy-
phenylalanine, beta-glutamic acid, beta-Hornoglutamic acid, or homoglutamic
acid;
Xaa at position 29 is Ile, Leu, Val, Ala, Phe, Asp, Glu, Lys, Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
or homoglutamic acid;
Xaa at position 30 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, Lys,
Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
or homoglutamic acid;
Xaa at position 31 is Trp, Phe, Tyr, Glu, Asp, or Lys;
Xaa at position 32 is Leu, Gly, Ala, Ser, Thr, Ile, Val, Glu, Asp, or Lys;
Xaa33 is: Val or Lys;
Xaa34 is: Lys or Asn;
Xaa36 is: Arg or Gly;
Xaa37 is: Gly or Pro;
wherein the compound has a GLP-1 activity.
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In the formulas listed herein, R1 can be a carboxyl group, an amine, an ester,
or a
substituted amine. Thus, the GLP-1 analogs as provided herein can have a
carboxyl or an
amide group at its C-terminal end.
A second family of GLP-1 analogs comprises or consists of the amino acid
sequence shown in formula II:
Xaa7-Xaa8-Xaa9-Xaa1 0-Xaa -Xaai 2-Xaa1 3-Xaa14-Xaa15-Xaa1 6-Xaa1 7-Xaa1
8_Xaa19-
Xaa20-Xaa21-Xaa22- Xaa23-Xaa24-Xaa25-Xaa26-Xaa27Aaa28-Xaa29-Xaa3o-Xaa3 -
Xaa32-Xaa33_Xaa34_Xaa35..Xaa36..Xaa37-Cys-Ser-G1y_Gly-C(0)-Ri (Formula II,
SEQ ID NO: 9)
wherein,
R1 is OR2 or NR2R3;
R2 and R3 are independently hydrogen or (Ci-C8)alkyl;
Xaa at position 7 is: L-histidine, D-histidine, desarnino-histidine, 2-amino-
histidine, 3-hydroxy-histidine, homohistidine, a-fluoromethyl-histidine or a-
methyl-histidine;
Xaa at position 8 is Gly, bAla (2-aminopropionic acid), 1-amino-
cylcopentanecarboxylic acid, 2-aminoisobutryic acid or an alpha-alpha-
disubstituted amino acid;
Xaa at position 9 is Glu, Asp, or Lys;
Xaa at position 10 is Gly or His;
Xaa at position 11 is Thr, Ala, Gly, Ser, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 12 is: His, Trp, Phe, or Tyr;
Xaa at position 13 is Thr or Gly;
Xaa at position 14 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 15 is Asp or Glu;
Xaa at position 16 is Val, Ala, Gly, Ser, Thr, Leu, Ile, Tyr, Glu, Asp, Trp,
or Lys;
Xaa at position 17 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 18 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, Trp, Tyr,
Lys,
Homolysine, Omithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-
Homoglutamic acid, or homoglutamic acid;
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Xaa at position 19 is Tyr, Phe, Trp, Glu, Asp, Gin, Lys, Homolysine,
Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or
homoglutamic acid;
Xaa at position 20 is Leu, Ala, Gly, Ser, Thr, Ile, Val, Glu, Asp, Met, Trp,
Tyr,
Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, or
homoglutamic acid;
Xaa at position 21 is Glu, Asp, Lys, Homolysine, Ornithine, 4-carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic
acid;
Xaa at position 22 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, Lys,
Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Hornoglutamic
acid,
or homoglutamic acid;
Xaa at position 23 is Gin, Asn, Arg, Glu, Asp, Lys, Ornithine, 4-carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic
acid;
Xaa at position 24 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Arg, Glu, Asp, Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-
Homoglutamic acid, or homoglutamic acid;
Xaa at position 25 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, Lys,
Homolysine,
Ornithine, 4-carboxy-phenyla1anine, beta-glutamic acid, beta-Homoglutamic
acid,
or homoglutamic acid;
Xaa at position 26 is Lys, Homolysine, Arg, Gin, Glu, Asp, His, Ornithine, 4-
carboxy-phenylalanine, beta-glutamic acid, or homoglutamic acid;
Xaa at position 27 is Leu, Glu, Asp, Lys, Homolysine, Ornithine, 4-carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic
acid;
Xaa at position 28 is Phe, Trp, Asp, Glu, Lys, Homolysine, Ornithine, 4-
carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic
acid;
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Xaa at position 29 is Ile, Leu, Val, Ala, Phe, Asp, Glu, Lys, Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
or homoglutamic acid;
Xaa at position 30 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, Lys,
Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
or homoglutamic acid;Xaa at position 31 is Trp, Phe, Tyr, Glu, Asp, or Lys;
Xaa at position 32 is Leu, Gly, Ala, Ser, Thr, Ile, Val, Glu, Asp, or Lys;
Xaa at position 33 is Val, Gly, Ala, Ser, Thr, Leu, Ile, Glu, Asp, or Lys;
Xaa at position 34 is Asn, Lys, Arg, Glu, Asp, or His;
Xaa at position 35 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 36 is Gly, Arg, Lys, Glu, Asp, or His;
Xaa at position 37 is Pro, Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, or
Lys;
wherein the compound has a GLP-1 activity.
A third family of GLP-1 analogs comprises or consists of the amino acid
sequence shown in formula formula III (SEQ ID NO: 13):
Xaa7-Xaa8-Xaa9-Xaa1 0-Xaa1 -Xaai 2-Xaa1 3-Xaa1 4-Xaa1 5-Xaa1 6-Xaa1 7-
Xaa13_Xaa19-
Xaa20-Xaa2 -Xaa22- Xaa23-Xaa24-Xaa25-Xaa26-Xaa27Aaa28-Xaa29-Xaa30Aaa3I-
Xaa32-Xaa33_Xaa34_Xaa35_Xaa36_Gly-Xaa38Aaa39_Xaa40..Xaa41- C(0)-R (Formula
III, SEQ ID NO: 13)
wherein,
R1 is OR2 or NR2R3;
R2 and R3 are independently hydrogen or (Ci-C8)alkyl;
Xaa at position 7 is: L-histidine, D-histidine, desamino-histidine, 2-amino-
histidine, 3-hydroxy-histidine, homohistidine, a-fluoromethyl-histidine or a-
methyl-histidine;
Xaa at position 8 is Gly, bAla (2-aminopropionic acid), 1-amino-
cylcopentanecarboxylic acid, 2-aminoisobutryic acid or an alpha-alpha-
disubstituted amino acid;
Xaa at position 9 is Glu, Asp, or Lys;
Xaa at position 10 is Gly or His;
Xaa at position 11 is Thr, Ala, Gly, Ser, Leu, Ile, Val, Glu, Asp, or Lys;
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Xaa at position 12 is His, Trp, Phe, or Tyr;
Xaa at position 13 is Thr or Gly;
Xaa at position 14 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 15 is Asp or Glu;
Xaa at position 16 is Val, Ala, Gly, Ser, Thr, Leu, Ile, Tyr, Glu, Asp, Trp,
or Lys;
Xaa at position 17 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 18 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, Trp, Tyr,
Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-
Homoglutamic acid, or homoglutamic acid;
Xaa at position 19 is Tyr, Phe, Trp, Glu, Asp, Gin, Lys, Homolysine,
Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or
homoglutamic acid;
Xaa at position 20 is Leu, Ala, Gly, Ser, Mr, Ile, Val, Glu, Asp, Met, Trp,
Tyr,
Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, or
homoglutamic acid;
Xaa at position 21 is Glu, Asp, Lys, Homolysine, Ornithine, 4-carboxy-
phenylalanine, beta-glutamic acid, beta-Homogiutamic acid, or homoglutamic
acid;
Xaa at position 22 is 2-aminoisobutyric acid, 1-amino-cylcopentanecarboxylic
acid, an alpha-alpha-disubstituted amino acid, or 2-aminoadipic acid;
Xaa at position 23 is Gin, Asn, Arg, Glu, Asp, Lys, Ornithine, 4-carboxy-
phenylalanine, beta-glutamic acid, beta-Homogiutamic acid, or homoglutamic
acid;
Xaa at position 24 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Arg, Glu, Asp, Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-
Homoglutamic acid, or homoglutamic acid;
Xaa at position 25 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, Lys,
Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
or homoglutamic acid;
Xaa at position 26 is Lys, Homolysine, Arg, Gin, Glu, Asp, His, Ornithine, 4-
carboxy-phenylalanine, beta-glutamic acid, or homoglutamic acid;
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Xaa at position 27 is Leu, Glu, Asp, Lys, Homolysine, Ornithine, 4-carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic
acid;
Xaa at position 28 is Phe, Trp, Asp, Glu, Lys, Homolysine, Ornithine, 4-
carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic
acid;
Xaa at position 29 is Ile, Leu, Val, Ala, Phe, Asp, Glu, Lys, Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
or homoglutamic acid;
Xaa at position 30 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, Lys,
Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
or homoglutamic acid;
Xaa at position 31 is Trp, Phe, Tyr, Glu, Asp, or Lys;
Xaa at position 32 is Leu, Gly, Ala, Ser, Thr, Ile, Val, Glu, Asp, or Lys;
Xaa at position 33 is Val, Gly, Ala, Ser, Thr, Leu, Ile, Glu, Asp, or Lys;
Xaa at position 34 is Asn, Lys, Arg, Glu, Asp, or His;
Xaa at position 35 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 36 is Gly, Arg, Lys, Glu, Asp, or His;
Xaa at position 38 is Cys, Gly, or is omitted;
Xaa at position 39 is Ala, Gly, Ser, Cys, or is omitted;
Xaa at position 40 is Gly or is omitted;
Xaa at position 41 is Gly or is omitted;
provided that when the amino acid at position 38, 39, 40, or 41 is omitted,
then each
amino acid downstream of that amino acid is also omitted, and wherein the
compound
has a GLP-1 activity. Thus, for example, if the amino acid at position 38 is
omitted, then
there are also no amino acids at positions 39-41. Similarly, if the amino acid
at position
39 is omitted, there there are also no amino acids at positions 40 and 41. And
if the
amino acid at position 40 is omitted, then there is no amino acid at position
41.
Another family of GLP-1 analogs includes a ring or cyclic structure that is
formed
when two amino acids within the analog are joined together, typically via
their side
chains. The side chains may be joined directly to one another or via a linker.
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Certain GLP-1 analogs in this family have the general structure shown in
Formula
V:
rm 0
X1 X2 _____________________________ X3 __ x4-x5 1_2
5 (Formula V)
wherein
X is ¨N(Z1)-C(0)- or ¨C(0)-N(Zi)-;
Z1 is hydrogen or (C1-C8)alkyl;
X1 is at least 11 amino acids;
X2 is an amino acid;
X3 is a bond or 1-5 amino acids;
X4 is an amino acid;
X5 is at least 10 amino acids;
Z2 is -0Z3 or --NZ4Z5; and
Z3, Z4, and Z5 are independently hydrogen or (C1-C8)alkyl;
and wherein the amino acids comprising X1 and X5 correspond to amino acids
from the
amino and carboxyl portions of GLP-1, respectively, or similar sequences in
which amino
acids in the native GLP-1 sequence have been substituted with an amino acid
such as
described in Formulas X to Y.
Some cyclic GLP-1 analogs, for instance, comprise or consist of the amino acid
sequence shown in formula IV:
Xaa.7-Xaa8-Xaa9-Xaa10-Xaal 1-Xaa1 2-Xaa1 3-Xaa1 4-Xaa15-Xaa1 6-Xaa1 7-Xaa1 8-
Xaa19-
Xaa20-Xaa2 -Xaa22-Xaa23-Xaa24-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29-Xaa30-Xaa3i-
Xaa32-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-Xaa38-Xaa39-Xaa40-Xaa41 C(0)-R1
(Formula IV, SEQ ID NO: 29)
wherein,
R1 is OR2 or NR2R3;
R2 and R3 are independently hydrogen or (Ci-C8)alkyl;
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Xaa at position 7 is: L-histidine, D-histidine, desamino-histidine, 2-amino-
histidine, 3-hydroxy-histidine, homohistidine, a-fluoromethyl-histidine or a-
methyl-histidine;
Xaa at position 8 is Gly, bAla (2-aminopropionic acid), 1-amino-
cylcopentanecarboxylic acid, 2-aminoisobutryic acid or an alpha-alpha-
disubstituted
amino acid;
Xaa at position 9 is Glu, Asp, or Lys;
Xaa at position 10 is Gly or His;
Xaa at position 11 is Thr, Ala, Gly, Ser, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 12 is: His, Trp, Phe, or Tyr;
Xaa at position 13 is Thr or Gly;
Xaa at position 14 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 15 is Asp or Glu;
Xaa at position 16 is Val, Ala, Gly, Ser, Thr, Leu, Ile, Tyr, Glu, Asp, Trp,
or Lys;
Xaa at position 17 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 18 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, Trp, Tyr,
Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-
Homoglutamic acid, alpha, gamma-diaminobutryic acid, or homoglutamic acid;
Xaa at position 19 is Tyr, Phe, Trp, Glu, Asp, Gin, Lys, Homolysine,
Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, alpha,
gamma-diaminobutryic acid, or homoglutamic acid;
Xaa at position 20 is Leu, Ala, Gly, Ser, Thr, Ile, Val, Glu, Asp, Met, Trp,
Tyr,
Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid,
alpha,
gamma-diaminobutryic acid, or homoglutamic acid;
Xaa at position 21 is Glu, Asp, Lys, Homolysine, Ornithine, 4-carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, alpha, gamma-
diaminobutryic acid, or homoglutamic acid;
Xaa at position 22 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, Lys,
Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
alpha, gamma-diaminobutryic acid, or homoglutamic acid;
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Xaa at position 23 is Gin, Asn, Arg, Glu, Asp, Lys, Ornithine, 4-carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, alpha, gamma-
diaminobutryic acid, or homoglutamic acid;
Xaa at position 24 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Arg, Glu, Asp, Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-
Homoglutamic acid, alpha, gamma-diaminobutryic acid, or homoglutamic acid;
Xaa at position 25 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, Lys,
Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
alpha, gamma-diaminobutryic acid, or homoglutamic acid;
Xaa at position 26 is Lys, Homolysine, Arg, Gin, Glu, Asp, His, Ornithine, 4-
carboxy-phenylalanine, beta-glutamic acid, alpha, gamma-diaminobutryic acid,
or
homoglutamic acid;
Xaa at position 27 is Leu, Glu, Asp, Lys, Homolysine, Ornithine, 4-carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, alpha, gamma-
diaminobutryic acid, or homoglutamic acid;
Xaa at position 28 is Phe, Trp, Asp, Glu, Lys, Homolysine, Ornithine, 4-
carboxy-
phenylalanine, beta-glutarnic acid, beta-Homoglutamic acid, alpha, gamma-
diaminobutryic acid, or homoglutamic acid;
Xaa at position 29 is Ile, Leu, Val, Ala, Phe, Asp, Glu, Lys, Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
alpha, gamma-diaminobutryic acid, or homoglutamic acid;
Xaa at position 30 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, Lys,
Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
alpha, gamma-diaminobutryic acid, or homoglutamic acid;
Xaa at position 31 is Trp, Phe, Tyr, Glu, Asp, or Lys;
Xaa at position 32 is Leu, Gly, Ala, Ser, Thr, Ile, Val, Glu, Asp, or Lys;
Xaa at position 33 is Val, Gly, Ala, Ser, Thr, Leu, Ile, Glu, Asp, or Lys;
Xaa at position 34 is Asn, Lys, Arg, Glu, Asp, or His;
Xaa at position 35 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 36 is Gly, Arg, Lys, Glu, Asp, or His;
Xaa at position 37 is Pro, Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, or
Lys;
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Xaa at position 38 is Gly, Ser, Lys, Cys, or is omitted;
Xaa at position 39 is Gly, Ala, Ser, Thr, Ile, Val, L,eu, Phe, Pro, Cys or is
omitted;
Xaa at position 40 is Gly, Cys, or is omitted;
Xaa at position 41 is Gly or is omitted;
wherein two amino acids selected from Xaa18, Xaa19, Xaa20, Xaa21, Xaa22,
Xaa23, Xaa24,
Xaa25, Xaa26, Xaa27, Xaa28, Xaa29, and Xaa30 are joined to form a ring and the
two amino
acids forming the ring are separated by 0, 1, 2, 3, 4 or 5 amino acids, and
wherein the
compound has a GL,P-1 activity.
In counting the number of amino acids involved in the separation, the two
amino
acids whose side chains are joined are excluded. Thus, for instance, the two
amino acids
whose side chains are joined to form a ring have 0 amino acids separating them
when the
two amino acids are beside each other (eg., when the amino acids at positions
Xaa18 and
Xaa19 are joined to form a ring).
In a particular embodiment, the two amino acids that are joined to form a ring
are
separated by 3 or 4 amino acids, including for example when:
Xaai8 is joined to either Xaa22 or Xaa23,; or
Xaa19 is joined to either Xaa23 or Xaa24; or
Xaa20 is joined to either Xaa24 or Xaa25 or
Xaa21 is joined to either Xaa25 or Xaa26,; or
Xaa22 is joined to eitherXaa26 or Xaa27; or
Xaa23 is joined to either Xaa27 or Xaa28; or
Xaa24 is joined to eitherXaa28 or Xaa29; or
Xaa25 is joined to either Xaa29 or Xaa30 or
Xaa26 is joined to either Xaa30 or Xaa3i =
A variety of different amino acids, amino acid analogs (e.g., unnatural amino
acids) can be inserted at these positions. The amino acid or analogs are
chosen to have
reactive functional groups in the side chain that can be reacted together
directly or via a
linker to form a ring. The ring in some cyclic analogs, for instance, is
formed by reacting
a carboxyl group in the side chain of one amino acid or analog with an amino
group in
the side chain of the second amino acid or analog to form a cyclic lactam.
Thus, for
example, in certain embodiments, a ring structure is formed between the side
chains of
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any two of Glu (E), Asp (D), Lys (K), Ornithine (0), 4-carboxy-phenylalanine
(Cpa),
beta-homoglutamic acid (B), alpha, gamma-diaminobutryic acid (Dab), and
homoglutamic acid (J) that are located at the positions specified above as
exemplified
below:
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O 0
N N
Asp-Lys Lys-Asp
O HN ---\ /----NH0
vw r
'--,
,
,
Glu-Lys Lys-Glu
O 0
iv H 1 1 H "7
Homo-Glu-Lys Lys-Homo-Glu
O 0
1
N'
.õ....--., .1 il N
Si
H
K-
1¨ 1
1
Cpa-Lys Lys-Cpa
O 0
N¨ N)
Asp-Orn Orn-Asp
H H
O N
Is' 1 I "7
Glu-Orn Orn-Glu
O 0
N NI)
Homo-Glu-Orn Orn-Homo-Glu
0 0
el 11\-11'¨ _¨N /10
H
----1 r---
-r
Cpa-Orn Orn-Cpa
O HN.--'-, ../-- NH 0
G
,
beta-Homo-Glu-Lys Lys-beta-Homo-Glu
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Figure 1, for example, depicts examples of the positions within GLP-1 that are
separated by 3 amino acids that can be joined to form a cyclic lactam.
Figures 2A through 21 provide chemical structures in which a glutamic
acid/lysine
amino acid pair are joined to form a ring. It will be appreciated by those
skilled in the art
that the particular pairing can be replaced with any of the other pairings
shown above, as
well as other pairings.
Specific examples of the sequences of GLP-1 analogs that have the requisite
amino acids at the appropriate locations that can be joined to form a cyclic
lactam are set
forth in SEQ ID NOS: 30 to 246 and are shown in Table 2, with the two amino
acids that
are joined to form the ring being indicated by bold and underlined type.
Some GLP-1 analogs include two cyclic structures. One example is
HGEGT FTSDV SSYLE GQAKK EFIAW LEKGR K (SEQ ID NO: 277)
In this analog, the first E and K pair (at positions 21 and 25, respectively)
form one cyclic
lactam and the second E and K pair (at positions 33 and 37, respectively) form
a second
cyclic lactam. One or both pairs of the Glu (E) and Lys (K) residues can be
substituted
with the other amino acids or amino acid analogs such as those listed and
illustrated
above to form the cyclic lactam rings.
A final family of GLP-1 analogs comprises or consists of the amino acid
sequence
shown in formula VI:
Xaa4-Xaa5-Xaa6-His7-A1a8-G1u9-G1y 0-Thr -Phe12-Thr1 3-S er 4-Asp's-Val 6-Ser
17-
Seri 8-Tyr 9-Leu20-G1u2 -Gly22-G1n2 3-Ala24-Ala25-Lys26-Glu27-Phe28-11e29-
Ala30-
Trp31-Leu32-Va133-Lys34-Gly35-Arg36-C(0)-R1 (Formula VI, SEQ ID NO: 276)
wherein,
R1 is OR2 or NR2R3;
R2 and R3 are independently hydrogen or (CI-C8)alkyl;
Xaa at position 4 is: Met or omitted;
Xaa at position 5 is: Met, His, or omitted;
Xaa at position 6 is: Met, Ala, Gly, Pro, Ser, Thr, Val, Gin, Arg, Lys, His,
Tyr,
Ile, Asp, Leu, Asn, Glu, Trp, or Phe;
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provided that when the amino acid at position 5 is omitted, then the amino
acid at
position 4 is also omitted, wherein the compound has a GLP-1 activity.
Certain GLP-1 analogs are exemplified in Table 2. Several of the sequences
listed in Table 2 are shown as having a C-terminal amide. It should be
understood,
however, that all of the sequences listed in the Table 2 and described
elsewhere in the
specification can have a C-terminal carboxyl group or a C-terminal amide
group. As
used in Table 2: bAla is beta-aminopropionic acid; Aad is 2-aminoadipic acid;
Z is Aib (2
aminoisobutyric acid); 0 is omithine; Cpa is 4-carboxy-phenylalanine; Dab is
alpha,
gamma-diaminobutryic acid, and B is beta homoglutamic acid; J is homoglutamic
acid.
[K /O/C] means that the amino acid sequence optionally comprises any one of
lysine,
ornithine or cysteine at this position, but may include none of these amino
acids.
Residues that are highlighted in bold and underlined indicate the amino acids
that are
joined (typically via their side chains) to form a ring or cyclic structure.
Table 2
GLP-1 Analog Internal Ref. SEQ
ID
NO:
HGEGT FTSDV SSYLE GQAAK EFIAW LVKGR G 2
MHGEGT FTSDV SSYLE GQAAK EFIAW LVKGR 3
HGEGT FTSDV SSYLE GQAAK EFIAW LVKGR GGGGG C cgGLP-11 4
Formula I 5
XGXXX XXXXX XXXXX XCXXX XXXXX XXXGX XSSGA PPPS
HGEGT FTSDV SSYLE GCAAK EFIAW LVKGR G cgGLP-1-3, 7, 9 6
HGEGT FTSDV SSYLE GCAAK EFIAW LVKGR GSSGA PPPS cgGLP-12, 14 7
HGEGT FTSDV SSYLE GCAAK EFIAW LKNGG PSSGA PPPS cgGLP-13, 15 8
XXXXX XXXXX XXXXX XXXXX XXXXX XXXXX XCSGG Formula II 9
HGEGT FTSDV SSYLE GQAAK EFIAW LVKGR GCSG cgGLP-4-6, 6, 10
10, 16
HZEGT FTSDV SSYLE GQAAK EFIAW LVKGR GCSGG cgGLP-17 11
H[bAla]EGT FTSDV SSYLE GQAAK EFIAW LVKGR GCSGG cgGLP-18 12
XXXXX XXXXX XXXXX XXXXX XXXXX XXXXX GXXXX Formula III 13
HAEGT FTSDV SSYLE ZQAAK EFIAW LVKGR - amide 14
HGEGT FTSDV SSYLE ZQAAK EFIAW LVKGR G - amide 15
HGEGT FTSDV SSYLE ZQAAK EFIAW LVKGR GC 16
HGEGT FTSDV SSYLE ZQAAK EFIAW LVKGR GC- amide cgGLP-23 17
HGEGT FTSDV SSYLE ZQAAK EFIAW LVKGR GCA-amide cgGLP30 and 18
mgGLP 20
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HGEGT FTSDV SSYLE ZQAAK EFIAW LVKGR GCG 19
HGEGT FTSDV SSYLE ZQAAK EFIAW LVKGR GCG- amide mgGLP-16 20
HGEGT FTSDV SSYLE ZQAAK EFIAW LVKGR GCSG cgGLP-19 21
HGEGT FTSDV SSYLE ZQAAK EFIAW LVKGR GCSG- amide cgGLP-24-29 22
mgGLP22, mgGLP-
33, mgGLP-27
HGEGT FTSDV SSYLE ZQAAK EFIAW LVKGR GCSGG 23
HGEGT FTSDV SSYLE ZQAAK EFIAW LVKGR GCSGG- amide mgGLP-15 24
HZEGT FTSDV SSYLE ZQAAK EFIAW LVKGR GCSG cgGLP-20 25
HZEGT FTSDV SSYLE ZQAAK EFIAW LVKGR GCSGG 26
HZEGT FTSDV SSYLE ZQAAK EFIAW LVKGR GGC cgGLP-22 27
HGEGT FTSDV SSYLE [Aad]QAAK EFIAW LVKGR GCSG cgGLP-21 28
Formula IV 29
HGEGT FTSDV SSYLE EQAAK EFIAW LVKGR G- amide cycloE22-K26 30
(mgGLP-24)
HGEGT FTSDV SSYLE EQAAK EFIAW LVKGR GCA- amide 31
HGEGT FTSDV SSYLE EQAAK EFIAW LVKGR GCSG-amide cycloE22- 32
K2G/CSG
HGEGT FTSDV SEYLE KQAAK EFIAW LVKGR G[K/O/C)-amide G8 cyclo [18- 33
22]
-Lys/Orn 38
HGEGT FTSDV SSELE GKAAK EFIAW LVKGR G[K/O/C]-amide G8 cyclo [19- 34
23]
-Lys/Orn 38
HGEGT FTSDV SSYEE GQKAK EFIAW LVKGR G[K/O/C]-amide [G1y8]GLP1(7- 35
37)
cyclo[G1u14-
Lys18]38
HGEGT FTSDV SSYLE GQAKK EFIAW LVKGR G[K/O/C]-amide [G1y8]GLP1(7- 36
37) cyc[G1u15-
Lys19]
HGEGT FTSDV SSYLE EQAAK EFIAW LVKGR G[K/O/C)-amide G8 cyclo (22- 37
26]
-Lys/Orn 38
HGEGT FTSDV SSYLE GEAAK KFIAW LVKGR G[K/O/C]-amide [G1y8]GLP1(7- 38
37)
cyclo[G1u17-
Lys21]
HGEGT FTSDV SSYLE GQEAK EKIAW LVKGR G[K/O/C]-amide [G1y8]GLP1(7- 39
37)
cyclo[G1u18-
Lys22]
HGEGT FTSDV SSYLE GQAEK EFKAW LVKGR G[K/O/C1-amide G8 cyclo [25- 40
29]
-Lys/Orn 38
HGEGT FTSDV SSYLE GQAAE EFIKW LVKGR G[K/O/C]-amide G8 cyclo [26- 41
30]
-Lys/Orn 38
HGEGT FTSDV SSYLE GQAAK EFIAW LVKGR G[K/O/C)-amide
[G1y8]cyclo[21- 42
26]GLP1(7-37)-
amide
HGEGT FTSDV SSYLE JQAAK EFIAW LVKGR G-amide cycloJ22-K26 43
HGEGT FTSDV SSYLE JQAAi EFIAW LVKGR GCA-amide cycloJ22-K26 44
HGEGT FTSDV SSYLE 77-'QAAi EFIAW LVKGR GCSG-amide cycloJ22- 45
K26/CSG
HGEGT FTSDV SJYLE KQAAK EFIAW LVKGR G[K/O/C]-amide G8 cyclo [18- 46
22]
-Lys/Orn/Cys 38
HGEGT FTSDV SSJLE GKAAK EFIAW LVKGR G[K/0/C]-amide GO cyclo [19- 47
23]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYJE GQRAK EFIAW LVKGR G[K/O/C]-amide G8 cyclo [20- 48
24]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLJ GQAKK EFIAW LVKGR G[K/O/C]-amide G8 cyclo [21- 49
25]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE JQAAK EFIAW LVKGR G[K/O/C]-amide G8 cyclo [22- 50
46
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26]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE GJAAK KFIAW LVKGR G[K/O/C]-amide G8 cyclo [23- 51
27]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE GQJAK EKIAW LVKGR G[K/O/C]-amide G8 cyclo [24- 52
28]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE GQAJK EFKAW LVKGR G[K/O/C)-amide G8 cyclo [25- 53
29)
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE GQAAJ EFIKW LVKGR GIK/0/C1-amide G8 cyclo [26- 54
30]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLJ GQAAK EFIAW LVKGR G(K/O/C)-amide G8 cyclo [21- 55
26]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE KQAAJ EFIAW LVKGR G-amide 56
HGEGT FTSDV SSYLE KQAAJ EFIAW LVKGR GCA-amide 57
HGEGT FTSDV SSYLE KQAAJ EFIAW LVKGR GCSG-amide 58
HGEGT FTSDV SKYLE JQAAK EFIAW LVKGR G[K/O/C)-amide 59
HGEGT FTSDV SSKLE GJAAK EFIAW LVKGR G[K/O/C]-amide 60
HGEGT FTSDV SS7KE GQJAK EFIAW LVKGR G[K/O/C)-amide 61
HGEGT FTSDV SSYLK GQAJK EFIAW LVKGR G[K/O/C)-amide 62
HGEGT FTSDV SSYLE KQAAJ EFIAW LVKGR G[K/O/C)-amide 63
HGEGT FTSDV SSYLE GKAAT JFIAW LVKGR G[K/O/C)-amide 64
HGEGT FTSDV SSYLE GQKAK EJIAW LVKGR G[K/O/C)-amide 65
HGEGT FTSDV SSYLE GQAKK ETJAW LVKGR G[K/O/C]-amide 66
HGEGT FTSDV SSYLE GQAAK EFTJW LVKGR G[K/O/C]-amide 67
HGEGT FTSDV SSYLK GQAAJ EFIAW LVKGR G[K/0/C1-amide 68
HGEGT FTSDV SSYLE EQAAO EFIAW LVKGR G-amide cycloE22-026 69
HGEGT FTSDV SSYLE EQAAO EFIAW LVKGR GCA-amide cycloE22-026 70
HGEGT FTSDV SSYLE EQAAO EFIAW LVKGR GCSG-amide cycloE22- 71
026/CSG
HGEGT FTSDV SEYLE OQAAK EFIAW LVKGR G[K/O/C]-amide G8 cyclo [18- 72
22]
-Lys/Orn/Cys 38
HGEGT FTSDV SSELE GOAAK EFIAW LVKGR G[K/O/C)-amide G8 cyclo [19- 73
23)
-Lys/Orn/Cys 38
HGEGT FTSDV SSYEE GQOAK EFIAW LVKGR G(K/0/C)-amide G8 cyclo [20- 74
24)
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE GQAOK EFIAW LVKGR G[K/O/C]-amide G8 cyclo [21- 75
25]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE EQAAO EFIAW LVKGR G[K/O/C)-amide G8 cyclo [22- 76
26]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE GEAAK OFIAW LVKGR G[K/O/C]-amide G8 cyclo [23- 77
27]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE GQEAK EOIAW LVKGR G[K/O/C)-amide G8 cyclo [24- 78
28)
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE GQAEK EFOAW LVKGR G[K/O/C]-amide G8 cyclo [25- 79
29]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE GQAAE EFIOW LVKGR G[K/O/C]-amide G8 cyclo [26- 80
30]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE GQAAO EFIAW LVKGR G[K/O/C)-amide G8 cyclo [21- 81
26]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE OQAAE EFIAW LVKGR G-amide 82
HGEGT FTSDV SSYLE -6-QAAT EFIAW LVKGR GCA-amide 83
HGEGT FTSDV SSYLE 0QAAi EFIAW LVKGR GCSG-amide 84
HGEGT FTSDV SOYLE EQAAT EFIAW LVKGR G[K/O/C1-amide 85
HGEGT FTSDV SOLE GEAAK EFIAW LVKGR G[K/O/C]-amide 86
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HGEGT FTSDV SSYOE GQEAK EFIAW LVKGR G[K/O/C)-amide 87
HGEGT FTSDV SSYLO GQAEK EFIAW LVKGR G[K/O/C]-amide 88
HGEGT FTSDV SSYLE OQAAE EFIAW LVKGR G[K/O/C]-amide 89
HGEGT FTSDV SSYLE GOAAT EFIAW LVKGR G[K/0/C1-amide 90
HGEGT FTSDV SSYLE GQOAK TEIAW LVKGR G[K/O/C]-amide 91
HGEGT FTSDV SSYLE GQAOK ETEAW LVKGR G[K/O/C]-amide 92
HGEGT FTSDV SSYLE GQAAO EFIEW LVKGR G[K/O/C]-amide 93
HGEGT FTSDV SSYLO GQAAE EFIAW LVKGR G[K/O/C]-amide 94
HGEGT FTSDV SSYLE DQAAO EFIAW LVKGR G-amide cycloD22-K26 95
HGEGT FTSDV SSYLE DQAAO EFIAW LVKGR GCA-amide cycloD22-K26 96
HGEGT FTSDV SSYLE DQAAO EFIAW LVKGR GCSG-amide cycloD22- 97
K26/CSG
HGEGT FTSDV SDYLE OQAAK EFIAW LVKGR G[K/O/C]-amide G8 cyclo [18- 98
22]
-Lys/Orn/Cys 38
HGEGT FTSDV SSDLE GOAAK EFIAW LVKGR G[K/O/C]-amide G8 cyclo [19- 99
23]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYDE GQOAK EFIAW LVKGR G[K/O/C]-amide G8 cyclo [20- 100
24]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLD GQAOK EFIAW LVKGR G[K/O/C]-amide G8 cyclo [21- 101
25]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE DQAAO EFIAW LVKGR G[K/O/C]-amide G8 cyclo [22- 102
26]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE GDAAK OFIAW LVKGR G[K/0/c]-amide G8 cyclo [23- 103
27]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE GQDAK EOIAW LVKGR G[K/O/C]-amide G8 cyclo [24- 104
28]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE GQADK EFOAW LVKGR G[K/O/C]-amide G8 cyclo [25- 105
29]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE GQAAD EFIOW LVKGR G[K/O/C]-amide G8 cyclo [26- 106
30]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE OQAAD EFIAW LVKGR G-amide 107
HGEGT FTSDV SSYLE OQAAD EFIAW LVKGR GCA-amide 108
HGEGT FTSDV SSYLE OQAAD EFIAW LVKGR GCSG-amide 109
HGEGT FTSDV SOYLE DQAAK EFIAW LVKGR G[K/0/C]-amide 110
HGEGT FTSDV SSOLE GDAAK EFIAW LVKGR G[K/O/C]-amide 111
HGEGT FTSDV SSYOE GQDAK EFIAW LVKGR G[K/O/C)-amide 112
HGEGT FTSDV SSYLO GQADK EFIAW LVKGR G[K/O/C]-amide 113
HGEGT FTSDV SSYLE OQAAD EFIAW LVKGR G[K/O/C]-amide 114
HGEGT FTSDV SSYLE GOAAT DFIAW LVKGR G[K/O/C]-amide 115
HGEGT FTSDV SSYLE GQOAK EDIAW LVKGR G[K/O/C]-amide 116
HGEGT FTSDV SSYLE GQAOK ETDAW LVKGR G[K/0/C]-amide 117
HGEGT FTSDV SSYLE GQAAO EFTDW LVKGR G[K/O/C]-amide 118
HGEGT FTSDV SSYLE GQAAO EFIAW LVKGR G[K/O/C]-amide G8 cyclo[2].-26] 119
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLO GQAAE EFIAW LVKGR G[K/O/C]-amide 120
HGEGT FTSDV SSYLE GQAAK EFIAW LVKGR G[K/O/C]-amide G8 cyclo[21-26] 121
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLK GQAAE EFIAW LVKGR G[K/O/C]-amide 122
HGEGT FTSDV SSYLJ GQAAK EFIAW LVKGR G[K/O/C]-amide 123
HGEGT FTSDV SSYLK GQA.Ari EFIAW LVKGR G[K/O/C]-amide 124
HGEGT FTSDV SSYLD GQAAO EFIAW LVKGR G[K/O/C]-amide 125
HGEGT FTSDV SSYLO GQAAT EFIAW LVKGR G[K/O/C]-amide 126
HGEGT FTSDV SSYLD GQAAR EFIAW LVKGR G[K/O/C]-amide 127
HGEGT FTSDV SSYLK GQAAD EFIAW LVKGR G[K/0/C]-amide 128
HGEGT FTSDV SSYL[Cpa] GQAAO EFIAW LVKGR G[K/O/C]-amide 129
HGEGT FTSDV SSYLO GQAA[Cpa] EFIAW LVKGR G[K/O/C]-amide , 130
HGEGT FTSDV SSYL[Cpa] GQAAK EFIAW LVKGR G[K/O/C]-amide 131
HGEGT FTSDV SSYLK GQAA[Cpa] EFIAW LVKGR G[K/O/C]-amide 132
HGEGT FTSDV SSYLB GQAAK EFIAW LVKGR G[K/O/C]-amide 133
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HGEGT FTSDV SSYLK GQAAB EFIAW LVKGR G[K/0/C]-amide 134
HGEGT FTSDV SSYLE DQAAK EFIAW LVKGR G-amide cycloD22-K26 135
HGEGT FTSDV SSYLE DQAAK EFIAW LVKGR GCA-amide cycloD22-K26 136
HGEGT FTSDV SSYLE DQAAE EFIAW LVKGR GCSG-amide cycloD22- 137
K26/CSG
HGEGT FTSDV SDYLE KQAAK EFIAW LVKGR G[K/0/C]-amide G8 cyclo [18- 138
22]
-Lys/Orn/Cys 38
HGEGT FTSDV SSDLE GKAAK EFIAW LVKGR G[K/O/C]-amide G8 cyclo [19- 139
23]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYDE GQKAK EFIAW LVKGR G[K/O/C)-amide G8 cyclo [20- 140
24]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLD GQAKK EFIAW LVKGR G[K/O/C]-amide G8 cyclo [21- 141
25]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE DQAAK EFIAW LVKGR G[K/O/C)-amide 08 cyclo [22- 142
26]
-Lys/Orn/Cys 36
HGEGT FTSDV SSYLE GDAAK KFIAW LVKGR G[K/O/C]-amide 08 cyclo [23- 143
27]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE GQDAK EKIAW LVKGR G[K/O/C]-amide G8 cyclo [24- 144
28]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE GQADK EFKAW LVKGR G[K/0/C1-amide G8 cyclo [25- 145
29]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE GQAAD EFIKW LVKGR G[K/O/CJ-amide G8 cyclo [26- 146
30]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE KQAAD EFIAW LVKGR G-amide 147
HGEGT FTSDV SSYLE 17QAAE EFIAW LVKGR GCA-amide 148
HGEGT FTSDV SSYLE EQAAD EFIAW LVKGR GCSG-amide 149
HGEGT FTSDV SKYLE EQAAK EFIAW LVKGR G[K/O/C]-amide 150
HGEGT FTSDV SSKLE GDAAK EFIAW LVKGR G[K/0/C1-amide 151
HGEGT FTSDV SSYKE GQDAK EFIAW LVKGR G[K/O/C1-amide 152
HGEGT FTSDV SSYLK GQTDK EFIAW LVKGR G[K/O/C]-amide 153
HGEGT FTSDV SSYLE KQATD EFIAW LVKGR G[K/0/C]-amide 154
HGEGT FTSDV SSYLE -GKAAK DFIAW LVKGR G[K/O/C]-amide 155
HGEGT FTSDV SSYLE GQKAK EDIAW LVKGR G[K/O/C]-amide 156
HGEGT FTSDV SSYLE GQTKK EFDAW LVKGR G[K/O/C]-amide 157
HGEGT FTSDV SSYLE GQATK EFTDW LVKGR G[K/O/C]-amide 158
HGEGT FTSDV SSYLE KQAAE EFIAW LVKGR G-amide GLP1(7-37) 159
cyclo[Lys16-
Glu20]
HGEGT FTSDV SSYLE KQAAE EFIAW LVKGR GCA-amide cycloK22-E26 160
HGEGT FTSDV SSYLE KQAAE EFIAW LVKGR GCSG-amide cyc1oK22- 161
E26/CSG
HGEGT FTSDV SKYLE EQAAK EFIAW LVKGR G[K/O/C]-amide 08 cyclo [18- 162
22]
-Lys/Orn/Cys 38
HGEGT FTSDV SSKLE GEAAK EFIAW LVKGR G[K/O/C]-amide 08 cyclo [19- 163
23]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYKE GQEAK EFIAW LVKGR G[K/O/C]-amide G8 cyclo [20- 164
24]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLK GQAEK EFIAW LVKGR G[K/O/C]-amide G8 cyclo [21- 165
25]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE KQAAE EFIAW LVKGR G[K/O/C1-amide G8 cyclo [22- 166
26]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE GKAAK EFIAW LVKGR G[K/O/C]-amide G8 cyclo [23- 167
27]
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE GQKAK EEIAW LVKGR G[K/O/C]-amide G8 cyclo [24- 168
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281
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE GQAKK EFEAW LVKGR G[K/O/C]-amide G8 cyclo [25- 169
29)
-Lys/Orn/Cys 38
HGEGT FTSDV SSYLE GQAAK EFIEW LVKGR G[K/O/C]-amide G8 cyclo [26- 170
30]
-Lys/Orn/Cys 38
HGEGT FTSDV SS[Cpa]LE GQAAK EFIAW LVKGR G[K/O/C]-amide G8 cyclo[19-
26] 171
-Lys/Orn/Cys 38
HGEGT FTSDV SSKLE GQAA[Cpa] EFIAW LVKGR G[K/O/C]-amide 172
HGEGT FTSDV SS[Cpa]LE GQAAO EFIAW LVKGR G[K/O/CI-amide G8 cyclo[19-
26I 173
-Lys/Orn/Cys 38
HGEGT FTSDV SSOLE GQAA[Cpa] EFIAW LVKGR G[K/O/C]-amide 174
HGEGT FTSDV SSYLE BQAAK EFIAW LVKGR G-amide 175
HGEGT FTSDV SSYLE TQAAK EFIAW LVKGR GCA-amide 176
HGEGT FTSDV SSYLE TQAAK EFIAW LVKGR GCSG-amide 177
HGEGT FTSDV SBYLE TQAAK EFIAW LVKGR G[K/O/C]-amide G8 cyclo [18- 178
22]
-Lys/Orn/Cys 38
HGEGT FTSDV SSBLE GKAAK EFIAW LVKGR G[K/O/C]-amide 179
HGEGT FTSDV SSYBE GQKAK EFIAW LVKGR G[K/O/C]-amide 180
HGEGT FTSDV SSYLB GQTKK EFIAW LVKGR G[K/O/C]-amide 181
HGEGT FTSDV SSYLE BQAAK EFIAW LVKGR G[K/O/C]-amide 182
HGEGT FTSDV SSYLE GBAAT KFIAW LVKGR G[K/O/C]-amide 183
HGEGT FTSDV SSYLE GQBAK TKIAW LVKGR G[K/O/C]-amide 184
HGEGT FTSDV SSYLE GQTBK EFKAW LVKGR G[K/O/C)-amide 185
HGEGT FTSDV SSYLE GQAAB EFTKW LVKGR G[K/O/C]-amide 186
HGEGT FTSDV SKYLE BQAAK EFIAW LVKGR G[K/O/C]-amide 187
HGEGT FTSDV SSYLE iQAAB EFIAW LVKGR G-amide 188
HGEGT FTSDV SSYLE TQAAB EFIAW LVKGR GCA-amide 189
HGEGT FTSDV SSYLE iQAAB EFIAW LVKGR GCSG-amide 190
HGEGT FTSDV SSKLE EBAAT EFIAW LVKGR G[K/O/C]-amide 191
HGEGT FTSDV SSYKE GQBAK EFIAW LVKGR G[K/O/C]-amide 192
HGEGT FTSDV SSYLK GQABK EFIAW LVKGR G[K/O/C]-amide 193
HGEGT FTSDV SSYLE KQAAB EFIAW LVKGR G[K/0/C1-amide 194
HGEGT FTSDV SSYLE GKAAK BFIAW LVKGR G[K/O/C]-amide 195
HGEGT FTSDV SSYLE GQKAK EBIAW LVKGR G[K/O/C]-amide 196
HGEGT FTSDV SSYLE GQAKK EFBAW LVKGR G[K/0/C]-amide 197
HGEGT FTSDV SSYLE GQAAK EFTBW LVKGR G[K/O/C)-amide 198
HGEGT FTSDV SSYLE [Cpa]QAAK ¨EFIAW LVKGR G-amide 199
HGEGT FTSDV SSYLE [Cpa]QAAK EFIAW LVKGR GCA-amide 200
HGEGT FTSDV SSYLE [Cpa]QAAK EFIAW LVKGR GCSG-amide 201
HGEGT FTSDV S[Cpa]YLE KQAAT E= FIAW LVKGR G[K/O/C)-amide 202
HGEGT FTSDV SS[Cpa]LE GKAAK EFIAW LVKGR G[K/O/C)-amide 203
HGEGT FTSDV SSY[Cpa]E GQKAK EFIAW LVKGR G[K/0/C1-amide 204
HGEGT FTSDV SSYL[Cpa] GQAKK EFIAW LVKGR G[K/O/C]-amide 205
HGEGT FTSDV SSYLE [Cpa]QAAK EFIAW LVKGR G[K/O/C]-amide 206
HGEGT FTSDV SSYLE G[Cpa]AA7 KFIAW LVKGR G[K/O/C]-amide 207
HGEGT FTSDV SSYLE GQ[Cpa]AK EKIAW LVKGR G[K/O/C]-amide 208
HGEGT FTSDV SSYLE GQA[Cpa]K ETKAW LVKGR G[K/O/C]-amide 209
HGEGT FTSDV SSYLE GQAA[Cpa] EFIKW LVKGR G[K/O/C)-amide 210
HGEGT FTSDV SKYLE [Cpa]QAAK EFIAW LVKGR G[K/0/C1-amide 211
HGEGT FTSDV SSYLE KQAA[Cpa] EFIAW LVKGR G-amide 212
HGEGT FTSDV SSYLE KQAA[Cpa] EFIAW LVKGR GCA-amide 213
HGEGT FTSDV SSYLE KQAA[Cpa] EFIAW LVKGR GCSG-amide 214
HGEGT FTSDV SSKLE E[Cpa]AAK EFIAW LVKGR G[K/O/C]-amide 215
HGEGT FTSDV SSKE GQ[Cpa]AK EFIAW LVKGR G[K/O/C]-amide 216
HGEGT FTSDV SSYTK GQA[Cpa]K EFIAW LVKGR G[K/O/C]-amide 217
HGEGT FTSDV SSYLE KQAA[Cpa] EFIAW LVKGR G[K/O/C]-amide 218
HGEGT FTSDV SSYLE GKAAK [Cpa]FIAW LVKGR G[K/O/C]-amide 219
HGEGT FTSDV SSYLE GQKAK E[Cpa]IAW LVKGR G[K/O/C]-amide 220
HGEGT FTSDV SSYLE GQTKK EF[Cpa]AW LVKGR G[K/O/C)-amide 221
HGEGT FTSDV SSYLE GQAAK EFI[Cpa]W LVKGR G[K/O/C]-amide 222
HGEGT FTSDV SSYLE [Cpa]QAA0 EFIAW LVKGR G-amide 223
HGEGT FTSDV SSYLE [Cpa]QAA0 EFIAW LVKGR GCA-amide 224
HGEGT FTSDV SSYLE [Cpa]QAAZ E= FIAW LVKGR GCSG-amide 225
HGEGT FTSDV S[CpalYLE 0QAAT E= FIAW LVKGR G[K/O/C]-amide 226
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HGEGT FTSDV SS[Cpa]LE GOAAK EFIAW LVKGR G[K/0/C]-amide 227
HGEGT FTSDV SSY[Cpa]E GQOAK EFIAW LVKGR G[K/O/C)-amide 228
HGEGT FTSDV SSYL[Cpa] GQ27,0K EFIAW LVKGR G[K/O/Cj-amide 229
HGEGT FTSDV SSYLE [Cpa]QAT,0 EFIAW LVKGR G[K/O/C)-amide 230
HGEGT FTSDV SSYLE G[Cpa]AAT OFIAW LVKGR G[K/O/C]-amide 231
HGEGT FTSDV SSYLE GQ[Cpa]AK EOIAW LVKGR G[K/O/C)-amide 232
HGEGT FTSDV SSYLE GQA[Cpa]K EFOAW LVKGR G[K/O/C]-amide 233
HGEGT FTSDV SSYLE GQAA[Cpa] EFIOW LVKGR G[K/O/C)-amide 234
HGEGT FTSDV SOYLE [Cpa]QAAK EFIAW LVKGR G[K/O/C)-amide 235
HGEGT FTSDV SSYLE 0QAA[Cpa] EFIAW LVKGR G-amide 236
HGEGT FTSDV SSYLE EQAA[Cpa] EFIAW LVKGR GCA-amide 237
HGEGT FTSDV SSYLE EQAA[Cpa] EFIAW LVKGR GCSG-amide 238
HGEGT FTSDV SSOLE E[Cpa]AAK EFIAW LVKGR G[K/O/C]-amide 239
HGEGT FTSDV SSEOE GQ[Cpa]AK EFIAW LVKGR G[K/0/C]-amide 240
HGEGT FTSDV SSYTO GQA[Cpa]K EFIAW LVKGR G[K/O/C)-amide 241
HGEGT FTSDV SSYLE 0QAA[Cpa] EFIAW LVKGR G[K/O/C]-amide 242
HGEGT FTSDV SSYLE aDAAK [Cpa]FIAW LVKGR G[K/O/C]-amide 243
HGEGT FTSDV SSYLE GQOAK E[Cpa]IAW LVKGR G[K/O/C]-amide 244
HGEGT FTSDV SSYLE GQEOK EF[Cpa]AW LVKGR G[K/O/C]-amide 245
HGEGT FTSDV SSYLE GQAAO EFI[Cpa]W LVKGR G[K/O/C)-amide 246
A HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR A-GLP-1 247
G HAEGT FTSDV SSYLE GQAAK
EFIAW LVKGR G-GLP-1 248
P HAEGT FTSDV SSYLE GQAAK
EFIAW LVKGR P-GLP-1 249
S HAEGT FTSDV SSYLE GQAAK
EFIAW LVKGR S-GLP-1 250
T HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR T-GLP-1 251
/ HAEGT FTSDV SSYLE GQAAK
EFIAW LVKGR , V-GLP-1 252
MO HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR MQ-GLP1 , 253
MR HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR MR-GLP1 254
MK HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR MK-GLP1 255
M HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR M-GLP1 256
MH HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR MH-GLP1 257
MHH HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR MHH-GLP1 258
MY HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR MY-GLP1 259
MI HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR MI-GLP1 260
MD HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR MD-GLP1 261
ML HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR ML-GLP1 262
MN HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR MN-GLP1 263
ME HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR ME-GLP1 264
MW HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR MW-GLP1 265
MF HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR MF-GLP1 266
MM HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR MM-GLP1 267
HGEGT FTSDV SSYLE GQAAK EFIAW LVKGR GCA -amide 268
HGEGT FTSDV SSYLE GQAAK EFIAW LVKGG G 269
HGEGT FTSDV SSYLE GQAAK EFIAW LKNGG G 270
HGEGT FTSDL SKQME EEAVR LFIEW LKNGG -amide 271
HGEGT FTSDL SKQME ZEAVR LFIEW LKNGG -amide 272
HGEGT FTSDV SSYLE GQAAK EFIAW L 273
HGEGT FTSDV SSYLE EQAKK EFIAW LVKGR G -amide 274
HGEGT FTSDV SSYLE EQAKK EFIAW LVKGR GCGS-amide 275
HGEGT FTSDV SSYLE GQAKK EFIAW LEKGR K-amide 277
HGEGT FTSDV SSYLE EQAADab EFIAW LVKGR G-amide 279
HGEGT FTSDV SSYLE EQAADab EFIAW LVKGR GCA-amide 280
HGEGT FTSDV SSYLE EQAADab EFIAW LVKGR GCSG-amide 281
HGEGT FTSDV SEYLE EabQAAK EFIAW LVKGR G[K/O/C]-amide 282
HGEGT FTSDV SSELE GDabAAK EFIAW LVKGR G[K/O/C]-amide 283
HGEGT FTSDV SSEEE GQDabAK EFIAW LVKGR G[K/O/C]-amide 284
HGEGT FTSDV SSYLE GQADabK EFIAW LVKGR G[K/O/C]-amide 285
HGEGT FTSDV SSYLE EQAADab EFIAW LVKGR G[K/O/C]-amide 286
HGEGT FTSDV SSYLE GEAAK DabFIAW LVKGR G[K/O/C]-amide 287
HGEGT FTSDV SSYLE GQEAK EDabIAW LVKGR G[K/O/C)-amide 288
HGEGT FTSDV SSYLE GQAEK EFDabAW LVKGR G[K/O/C]-amide , 289
HGEGT FTSDV SSYLE GQA-A-E EFIDabW LVKGR G[K/O/C]-amide 290
HGEGT FTSDV SSYLE GQAADab EFIAW LVKGR G[K/O/C]-amide 291
HGEGT FTSDV SSYLE DabQAAE EFIAW LVKGR G-amide 292
HGEGT FTSDV SSYLE DabQAAE EFIAW LVKGR GCA-amide 293
HGEGT FTSDV SSYLE DabQAAE EFIAW LVKGR GCSG-amide 294
HGEGT FTSDV SDabYLE EQAAT EFIAW LVKGR G[K/O/C]-amide 295
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HGEGT FTSDV SSDabLE GEAAK EFIAW LVKGR G[K/O/C]-amide 296
HGEGT FTSDV SSYDabE GQEAK EFIAW LVKGR G[K/O/C]-amide 297
HGEGT FTSDV SSYLDab GQAEK EFIAW LVKGR G[K/O/C]-amide 298
HGEGT FTSDV SSYLE DabQAAE EFIAW LVKGR G[K/O/C]-amide 299
HGEGT FTSDV SSYLE GDabAAK EFIAW LVKGR G[K/O/C]-amide 300
HGEGT FTSDV SSYLE GQDabAK EEIAW LVKGR G[K/O/C]-amide 301
HGEGT FTSDV SSYLE GQADabK EFEAW LVKGR G[K/O/C1-amide 302
HGEGT FTSDV SSYLE GQAADab EFIEW LVKGR G[K/O/CJ-amide 303
HGEGT FTSDV SSYLDab GQAAE EFIAW LVKGR G[K/O/C]-amide 304
The term "GLP-1 analog" also includes variants, fragments and derivatives of
the
foregoing GLP-1 analogs that are functional equivalents to one of the
foregoing GLP-1
analogs in that the variant, fragment or derivative has a similar amino acid
sequence (e.g.
comprising conservative substitutions) and retains, to some extent, at least
one activity of
the GLP-1 analog.
"GLP-1 variants" include polypeptides that are "substantially identical" (see
definition supra) to the GLP-1 analogs in the families listed above and in
Table 2. Such
variants include proteins having amino acid alterations such as deletions,
insertions
and/or substitutions. Typically, such alterations are conservative in nature
(see, e.g.,
Creighton, 1984, Proteins, W.H. Freeman and Company and discussion supra) such
that
the activity of the variant polypeptide is substantially similar to one of the
GLP-1 analogs
that are disclosed herein (e.g., has a GLP-1 activity such as insulinotropic
activity). In
the case of substitutions, the amino acid replacing another amino acid usually
has similar
structural and/or chemical properties. Insertions and deletions relative to
the GLP-1
analogs that are listed in the families above are typically involve 1, 2, 3,
4, 5, 6, 7, 8, 9 or
10 amino acids. In other instances, there are 1, 2, 3, 4 or 5 amino acid
insertions or
deletions.
A GLP-1 variant can have at least 75%, preferably at least 85%, more
preferably
at least 90%, 95%, 96%, 97%, 98%, or 99% amino acid identity with a GLP-1
analog as
described herein (i.e., the analogs in the different families listed above and
those listed in
Table 2), provided the variant still has a GLP-1 activity. For example, a
variant can have
at least 75%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid identity with a
GLP-1
analog of SEQ ID NO:7 or 8, provided that amino acid Xaa8, Xaa23, or any of
Xaa38 to
Xaa45 are unaltered. Other variants have at least 75%, 85%, 90%, 95%, 96%,
97%, 98%,
or 99% amino acid identity with a GLP-1 analog of SEQ ID NO:11 or 12, provided
that
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amino acid Xaa8 or any of Xaa38 to Xaa41 are unaltered. Certain other variants
have at
least 75%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid identity with a GLP-
1
analog of any one of SEQ ID NO:16-28, provided that amino acid Xaa8, Xaa22, or
any of
Xaa38 to Xaa41 when present, are unaltered. Still other variants have at least
75%, 85%,
90%, 95%, 96%, 97%, 98%, or 99% amino acid identity with a GLP-1 analog of any
one
of SEQ ID NO: 30 to 246 provided that two amino acids selected from Xaa.18,
Xaa19,
Xaa20, Xaa21, Xaa22, Xaa23, Xaa24, Xaa25, Xaa26, Xaa27, Xaa28, Xaa29, and
Xaa30 are joined
to form a ring. Still other variants have at least 75%, 85%, 90%, 95%, 96%,
97%, 98%,
or 99% amino acid identity with a GLP-1 analog of any one of SEQ ID NO: 247 to
267
with no more than 1, 2, 3, 4, or 5 conservative amino acid substitutions,
provided that the
amino acids Xaa4, Xaa5, or Xaa6, when present, are unaltered. Still other
variants have at
least 75%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid identity with a GLP-
1
analog of any one of SEQ ID NO: 268 to 275 with no more than 1, 2, 3, 4, or 5
conservative amino acid substitutions.
In certain embodiments, a GLP-1 variant comprises SEQ ID NO:7 or 8 with no
more than 1, 2, 3, 4 or 5 conservative amino acid substitutions, provided that
the
conservative amino acid substitutions are not at amino acid Xaa8, Xaa23, or
any of Xaa38 to
Xaa45 and the variant has a GLP-1 activity (e.g., insulinotropic activity).
In certain embodiments, a GLP-1 variant comprises SEQ ID NO:11 or 12 with no
more than 1, 2, 3, 4 or 5 conservative amino acid substitutions, provided that
the
conservative amino acid substitutions are not at amino acid Xaa8 or any of
Xaa38 to Xaa41
and the variant has a GLP-1 activity (e.g., insulinotropic activity).
In certain embodiments, a GLP-1 variant comprises any one of SEQ ID NO: 16-
28 with no more than 1, 2, 3, 4, or 5 conservative amino acid substitutions,
provided that
the conservative amino acid substitutions are not at amino acid Xaa8, Xaa22,
or any of
Xaa38 to Xaa4i.
In certain embodiments, a GLP-1 variant comprises any one of SEQ ID NO: 30 to
246 with no more than 1, 2, 3, 4 or 5 conservative amino acid substitutions,
provided that
two amino acids selected from Xaa18, Xaa19, Xaa20, Xaa2i, Xaa22, Xaa23, Xaa24,
Xaa25,
Xaa26, Xaa27, Xaa28, Xaa29, and Xaay, are joined to form a ring.
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In certain embodiments, a GLP-1 variant comprises any one of SEQ ID NO: 247
to 267 with no more than 1, 2, 3, 4, or 5 conservative amino acid
substitutions, provided
that the conservative amino acid substitutions are not at amino acid Xaa4,
Xaas, or Xaa6.
A "GLP-1 derivative" as used herein refers to one of the GLP-1 analogs listed
in
the families above and in Table 2 in which one or more amino acids has been:
1)
substituted with the corresponding D-amino acid, 2) altered to a non-naturally
occurring
amino acid residue, and/or 3) chemically modified. Examples of chemical
modification
include, but are not limited to alkylation, acylation, deamidation,
esterification,
phosphorylation, and glycosylation of the peptide backbone and/or amino acid
side
chains.
A "GLP-1 fragment" refers to truncated forms of the GLP-1 analogs listed in
the
families above or in Table 2 or variants or derivatives thereof. The fragments
typically
are truncated by 1, 2, 3, 4 or 5 amino acids relative to the GLP-1 analogs set
forth in the
families above. Truncation can be at either the amino and/or carboxyl
terminus.
GLP-1 compounds as provided herein can be complexed with suitable divalent
metal cations. Divalent metal complexes of GLP-1 compounds as provided herein
can be
administered subcutaneously as suspensions, and have a decreased rate of
release in vivo,
because such complexes of GLP-1 compounds as provided herein are generally
insoluble
in aqueous solutions of about physiological pH. Non-limiting examples of
divalent metal
cations suitable for complexing with a GLP-1 compound as provided herein
include Zn++,
Mn++,
Fe, Ca, Co, Cd++, Ni++, and the like. Divalent metal complexes of GLP-1
compounds as provided herein can be obtained, for example, using techniques as
described in WO 01/98331.
The GLP-1 compounds that are provided may simply comprise a GLP-1 analog as
disclosed herein or include an additional component, typically chosen to
extend the half-
life of the analog in vivo. Some GLP-1 compounds, for instance, are pegylated
to extend
the half-life of the molecule and/or reduce clearance. Other GLP-1 analogs are
modified
with a water-soluble polymer other than PEG. Suitable water-soluble polymers
or
mixtures thereof include, but are not limited to, N-linked or 0-linked
carbohydrates,
sugars (e.g. various polysaccharides such as chitosan, xanthan gum, cellulose
and its
derivatives, acacia gum, karaya gum, guar gum, carrageenan, and agarose),
phosphates,
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polyethylene glycol (PEG) (including the forms of PEG that have been used to
derivatize
proteins, including mono-(CI-C1o), alkoxy-, or aryloxy-polyethylene glycol),
monomethoxy-polyethylene glycol, dextran (such as low molecular weight dextran
of, for
example, about 6 kD), cellulose, or other carbohydrate based polymers, poly-(N-
vinyl
pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene
oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol),
polyoxyethylene-polyoxypropylene, polyvinyl alcohol, and copolymers of the
foregoing.
The GLP-1 compound, for instance, can include various components chosen to
increase
the in vivo half-life of the GLP-1 analog.
Yet another option is to fuse the GLP-1 analog to another polypeptide or
polypeptide domain. Thus, the GLP-1 compound can be a fusion protein in which
the
GLP-1 analogs disclosed herein are fused to various proteins such as the Fc
region of an
immunoglobulin, transferrin, or a blood component such as serum albumin (e.g.,
human
serum albumin), or fragments of these proteins. Exemplary amino acid sequences
for
human albumin are discussed in Lawn et al., 1981, Nucleic Acids Research
9:6102-6114;
Meloun et al., 1975, FEBS Lett. 58:136; and Minghetti et al., 1986, J Biol.
Chem.
261:6747). Such fusion proteins can be prepared using standard recombinant
techniques
such as those described herein and as known in the art.
Fusions can be made either at the amino-terminus, at the carboxyl-terminus of
the
GLP-1 analog or at both terminii. Fusions may be direct with no linker or
adapter
molecule or may be through a linker or adapter molecule. A linker or adapter
molecule
may be one or more amino acid residues, typically from about 20 to about 50
amino acid
residues. A linker or adapter molecule may also be designed with a cleavage
site for a
DNA restriction endonuclease or for a protease to allow for the separation of
the fused
moieties. It will be appreciated that once constructed, the fusion
polypeptides can be
derivatized according to the methods described herein.
B. Pegylation
In certain embodiments, a GLP-1 analog as provided herein is pegylated. As
used
herein, the terms "pegylated" and "pegylation" have their general meaning in
the art and
refer generally, for example, to the process of chemically modifying a GLP-1
analog as
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described herein by covalent attachment of one or more molecules of
polyethylene glycol
or a derivative thereof, such as by reacting a polyalkylene glycol, preferably
an activated
polyalkylene glycol, with a suitable reactive group or moiety such as an amino
acid, e.g.
lysine, to form a covalent bond. Although "pegylation" is often carried out
using
polyethylene glycol or derivatives thereof, such as methoxy polyethylene
glycol, the term
as used herein also includes any other useful polyalkylene glycol, such as,
for example
polypropylene glycol. As used herein, the term "PEG" refers to polyethylene
glycol and
its derivatives as understood in the art (see for example US Patent Nos:
5,445,090,
5,900,461, 5,932,462, 6,436,386, 6,448,369, 6,437,025, 6,448,369, 6,495,659,
6,515,100,
and 6,514,491).
GL,P-1 analogs as provided herein can be pegylated at random positions within
the peptide, or at predetermined positions within the molecule and can include
one or
more attached molecules, typically one, two, three, four, or five molecules.
The polymer used for peglylation can be of any molecular weight, and can be
branched or unbranched. For polyethylene glycol, generally the molecular
weight is
between about 1 kDa and about 100 kDa (the term "about" indicating that in
preparations
of polyethylene glycol, some molecules will weigh more, some less, than the
stated
molecular weight) for ease in handling and manufacturing. For example, the
polyethylene glycol can have an average molecular weight of about 1, 5, 10,
15, 20, 25,
30, 35, 40, 50, 60, 70, 80, 90 or 100 kDa. Some GL,P-1 compounds are pegylated
with
one or more molecules that are less than 40, 30 or 20 kDa. Other sizes can be
used,
depending on the desired therapeutic profile (e.g., the duration of
circulating half-life
desired, the effects, if any on biological activity, the ease in handling, the
degree or lack
of antigenicity and other known effects of the polyethylene glycol to a
therapeutic protein
or analog).
The PEG molecules (or other water-soluble polymers described herein) should be
attached to the GLP-1 analogs with consideration of effects on functional or
antigenic
domains of the polypeptides or proteins. For example, PEG can be covalently
bound
through amino acid residues via a reactive group, such as, a free amino,
carboxyl group
or sulfhydryl group. Reactive groups are those to which an activated PEG
molecule can
be bound. Examples of naturally occuring amino acid residues having a free
amino
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group include lysine residues and the N-terminal amino acid residues; those
having a free
carboxyl group include aspartic acid residues glutamic acid residues and the C-
terminal
amino acid residue. Sulfhydryl groups (e.g., on cysteine) can also be used as
a reactive
group for attaching the polyethylene glycol molecules. PEG molecules may also
be
incorporated by conjugation to reactive functional groups introduced
synthetically as
unnatural amino acids or alternatively, PEG may be conjugated to the peptide
using
orthogonal methods during peptide synthesis.
A variety of strategies can be used for pegylation of a GLP-1 analog (see,
e.g.,
WO 2005/042027, WO 2004/060386, Veronese, 2001, Biomaterials 22:405-417;
Roberts
et al., 2002, Advanced Drug Delivery Reviews 54:459-476; see also EP 0 401 384
(coupling PEG to G-CSF), and Malik et al., 1992, Exp. Hematol. 20:1028-1035
(reporting pegylation of GM-CSF using tresyl chloride). For example, PEG can
be
linked to GLP-1 analogs via covalent bonds to lysine, histidine, aspartic
acid, glutamic
acid, or cysteine residues. One or more reaction chemistries can be employed
to attach
polyethylene glycol to specific amino acid residues (e.g., lysine, histidine,
aspartic acid,
glutamic acid, or cysteine) of the GLP-1 analog or to more than one type of
amino acid
residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and
combinations
thereof) of the GLP-1 analog.
One such strategy is to link a PEG to a cysteine that is part of the GLP-1
analog.
As shown supra, several of the GLP-1 analogs that are provided include a
cysteine
residue at or near the C-terminus at which PEG can be attached. Attachment to
cysteine
can be achieved using various approaches. One common method involves reacting
a
PEG-maleimide to the thiol group of cysteine.
Another approach is to attach PEG to the carboxy-terminus of the GLP-1 analog
via enzymatic coupling (see, e.g., U.S. Patent No. 4,343,898).
PEG can be attached to the GLP-1 analog either directly or by an intervening
linker. Linkerless systems for attaching polyethylene glycol to proteins and
polypeptides
are described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-
304, 1992;
Francis et al., 1998, Intern, J. of Hematol. 68:1-18; U.S. Pat. No. 4,002,531;
U.S. Pat.
No. 5,349,052; WO 95/06058; and WO 98/32466.
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One method for attaching PEG directly to amino acid residues of GLP-1 analogs
without an intervening linker employs tresylated MPEG, which is produced by
the
modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride
(C1S02CH2CF3). Upon reaction of the protein or polypeptide with tresylated
MPEG,
polyethylene glycol is directly attached to amine groups of the protein or
polypeptide.
Thus, GLP-1 analog-PEG conjugates can be produced by reacting GLP-1 analogs
with a
PEG molecule having a 2,2,2-trifluoreothane sulphonyl group.
PEG can also be attached to GLP-1 analogs using a number of different
intervening linkers. For example, U.S. Pat. No. 5,612,460 discloses urethane
linkers for
connecting PEG to GLP-1 analogs. GLP-1 analog-PEG conjugates wherein the PEG
is
attached to the GLP-1 analog by a linker can also be produced by reaction of
GLP-1
analogs with compounds such as MPEG-succinimidylsuccinate, MPEG activated with
1,1'-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate,
MPEG-.rho.-
nitrophenolcarbonate, and various MPEG-succinate derivatives. A number of
additional
polyethylene glycol derivatives and reaction chemistries for attaching
polyethylene
glycol to proteins and polypeptides are described in WO 98/32466.
The number of polyethylene glycol moieties attached to each GLP-1 analog
(i.e.,
the degree of substitution) can also vary. For example, the pegylated GLP-1
analogs can
be linked, on average, to 1, 2, 3, 4, or 5, or more polyethylene glycol
molecules.
Methods for determining the degree of substitution are discussed, for example,
in
Delgado et al., 1992, Crit. Rev. Thera. Drug Carrier Sys. 9:249-304.
To effect covalent attachment of PEG to a GLP-1 analog, the hydroxyl end
groups
of the PEG must first be converted into reactive functional groups. This
process is
frequently referred to as "activation" and the product is called "activated
PEG." Methoxy
polyethylene glycol (mPEG), distally capped with a reactive functional group
is often
used. One such activated PEG is succinimidyl succinate derivative of PEG (SS-
PEG).
See also Abuchowski et al., 1984, Cancer Biochem. Biophys. 7:175-186; and U.S.
Pat.
No. 5,122,614 which discloses polyethylene glycol-N-succinimide carbonate and
its
preparation.
The Example section below provides detailed guidance on strategies for
pegylating the GLP-1 compounds that are disclosed herein.
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IV. Nucleic Acids and Methods of Preparing the GLP-1 Compounds and Analogs
The GLP-1 analogs that are provided can be produced using various methods that
are established in the art, including chemical synthesis and/or recombinant
methods.
Different strategies for attaching PEG to the peptide have been set forth
above.
If the GLP-1 analog is prepared by chemical synthesis, such methods typically
involve solid-state approaches, but can also utilize solution-based
chemistries or
combinations of solid-state and solution approaches. The Example section below
includes detailed guidance on the synthesis of the GLP-1 compunds described
herein,
including the various cyclic compounds that include a ring.
Examples of solid-state methodologies for synthesizing proteins are described
by
Merrifield, 1964, J. Am. Chem. Soc. 85:2149; and Houghton, 1985, Proc. Natl.
Acad. Sci.
825132. Fragments of the GLP-1 analog can also be synthesized and then joined
together. Methods for conducting such reactions are described by Grant, 1992,
Synthetic
Peptides: A User Guide, W.H. Freeman and Co., N.Y.; and in "Principles of
Peptide
Synthesis," 1993 (Bodansky and Trost, ed.), Springer-Verlag, Inc. N.Y. Further
guidance
=
on methods for preparing peptides sufficient to guide the skilled practitioner
in the
preparation of the GLP-1 analogs described herein is provided by: Liu et al.,
1996, J.
Am. Chem. Soc. 118:307-312; KuMann, 1987, Enzymatic Peptide Synthesis, CRC
Press,
Boca Raton, FL, pp. 41-59; Dryland et al., 1986, J. Chem. Soc., Perkin Trans.
1:125-137;
Jones, 1991, The Chemical Synthesis of Peptides, Clarendon Press; and
Bodanszky, M.
and Bodanszky A., 1994, The Practice of Peptide Synthesis, 2"d Ed., Springer-
Verlag).
Alternatively, the GLP-1 analogs can be prepared using established recombinant
techniques. For example, a GLP-1 analog can be expressed in a host cell by
introducing
into the cell a recombinant nucleic acid construct encoding a GLP-1 analog.
According
to such embodiments, the cells are transformed with the recombinant nucleic
acid
construct using any method for introducing polynucleotides into a host cell,
including, for
example packaging the polynucleotide in a virus (or into a viral vector) and
transducing a
host cell with the virus (or vector), or by transfection procedures known in
the art, as
exemplified by U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455.
The transformation procedure
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used may depend upon the cell to be transformed. Methods for introduction of
heterologous polynucleotides into mammalian cells are well known in the art
and include,
but are not limited to, dextran-mediated transfection, calcium phosphate
precipitation,
polybrene mediated transfection, protoplast fusion, electroporation,
encapsulation of the
polynucleotide(s) in liposomes, mixing nucleic acid with positively-charged
lipids, and
direct microinjection of the DNA into cells and cell nuclei.
A nucleic acid molecule encoding all or a functional portion of the GLP-1
analog
amino acid sequence can be inserted into an appropriate expression vector
using
conventional recombinant genetic techniques. The vector is typically selected
to be
functional in the particular host cell employed (i.e., the vector is
compatible with the host
cell machinery, permitting amplification and/or expression of the gene). For a
review of
expression vectors, see Nolan and Shatzman, 1998, Curr. Opin. Biotechnol.
9:447-450.
Expression vectors may be constructed from a convenient starting vector such
as
a commercially available vector. Such vectors may or may not contain all of
the desired
flanking sequences. Where one or more of the flanking sequences described
herein are
not already present in the vector, they may be individually obtained and
ligated into the
vector. Methods used for obtaining each of the flanking sequences are well
known to one
skilled in the art.
After the vector has been constructed and a nucleic acid molecule encoding GLP-
1 analog has been inserted into the proper site of the vector, the completed
vector may be
inserted into a suitable host cell for amplification and/or polypeptide
expression. The
transformation of an expression vector encoding GLP-1 analog into a selected
host cell
may be accomplished by well-known methods including methods such as
transfection,
infection, calcium chloride, electroporation, microinjection, lipofection,
DEAE-dextran
method, or other known techniques as described above. The method selected will
in part
be a function of the type of host cell to be used. These methods and other
suitable
methods are well known to the skilled artisan, and are set forth, for example,
in
Sambrook et al., 2001, MOLECULAR CLONING: A LABORATORY MANUAL, 3d
ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
A host cell, when cultured under appropriate conditions, synthesizes a GLP-1
analog that can subsequently be collected from the culture medium (if the host
cell
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secretes it into the medium) or directly from the host cell producing it (if
it is not
secreted) if collection of the protein is desired. Selection of an appropriate
host cell will
depend upon a number of different factors, such as desired expression levels,
polypeptide
modifications that are desirable or necessary for activity (such as
glycosylation or
phosphorylation) and ease of folding into a biologically-active molecule.
Mammalian cell lines available as host cells for expression are well known in
the
art and include, but are not limited to, many immortalized cell lines
available from the
American Type Culture Collection (ATCC), such as Chinese hamster ovary (CHO)
cells,
HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human
hepatocellular carcinoma cells (e.g., Hep G2), and a number of other cell
lines. In certain
embodiments, cell lines may be selected through determining which cell lines
have high
expression levels of GLP-1 analog.
V. Exemplary Therapeutic Utilities
In view of the various activities associated with GLP-1 (see Background), the
GLP-1 compounds that are described herein can be used generally to achieve one
or more
of the following biological activities: 1) stimulate insulin release, 2)
reduce blood
glucose levels, 3) increase plasma insulin levels, 4) stimulate transcription
of 13-cell-
specific genes (e.g., GLUT-1 transporter, insulin receptor and hexokinase-1),
5) increase
13-cell mass by inhibiting 13-cell apoptosis and increasing f3-cell
proliferation and
replication, 6) induce satiety thereby reducing food intake and promoting
weight loss, 7)
reduce gastric secretion, 8) delay gastric emptying, and 9) reduce gastric
motility.
The GLP-1 compounds can thus be used to treat a number of different forms of
diabetes or diseases closely related thereto, including but not limited to,
diabetes mellitus
of Type I or Type II, impaired glucose tolerance, insulin resistance, latent
autoimmune
diabetes Adult (LADA), gestational diabetes, metabolic syndrome, and maturity-
onset
diabetes of the young (MODY). Thus, the GLP-1 compounds can be used to treat
individuals having decreased sensitivity to insulin due to infection, stress,
stroke, or due
to a decreased sensitivity induced during pregnancy. Other types of diabetes
that can be
treated are those in which diabetes is linked to another endocrine disease
such as
glucagonoma, primary aldosteronism, Cushing's syndrome and somatostatinoma, or
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diabetes that arises due to administration of certain drugs or hormones (e.g.,
estrogen-
containing pharmaceuticals, psychoactive drugs, antihypertensive drugs, and
thiazide
diuretics).
The GLP-1 compounds can also be used to treat various coronary diseases and
diseases associated with lipid disorders, including, for instance,
hypertension, coronary
artery disease, hyperlipidemia, cardiovascular disease, atherosclerosis and
hypercholesteremia and myocardial infarction.
Bone disorders, osteoporosis and other related diseases can also be treated
with
the GLP-1 compositions.
Additional diseases that can be treated with the GLP-1 compounds include:
obesity, irritable bowel syndrome, stroke, catabolic changes after surgery,
myocardial
infarction,), and hyperglycemia. The GLP-1 compounds can also be used as a
sedative.
The GLP-1 compounds can also be used prophylactically, including treating
individuals at risk for developing a disease such as listed above. As a
specific example,
the compounds can be administered prophylactially to an individual at risk for
non-
insulin dependent diabetes or becoming obese. Such individuals include, for
instance,
those that have impaired glucose tolerance, those that are overweight and
those with a
genetic predisposition to the foregoing diseases (e.g., individuals from
families with a
history of diabetes).
A variety of different subjects can be treated with the GLP-1 compounds. The
term "subject" or "patient" as used herein, typically refers to a mammal, and
often, but
not necessarily, is a human that has or is at risk for one of the foregoing
diseases. The
subject, however, can also be a non-human primate (e.g., ape, monkey, gorilla,
chimpanzee). The subject can also be a mammal other than a primate such as a
veterinarian animal (e.g., a horse, bovine, sheep or pig), a domestic animal
(e.g., cat or
dog) or a laboratory animal (e.g., mouse or rat).
VI. Pharmaceutical Compositions
A. Composition
The GLP-1 compounds that are provided herein can be used as the active
ingredient in pharmaceutical compositions formulated for the treatment of the
diseases
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listed in the section on therapeutic utilities. Thus, the GLP-1 compounds that
are
disclosed can be used in the preparation of a medicament for use in various
therapeutic
applications, including those listed supra.
In addition to the GLP-1 compound, pharmaceutical compositions can also
include one or more other therapeutic agents that are useful in treating one
or more the
various disorders for which the GLP-1 compounds have utility. General classes
of other
therapeutic agents that can be combined with certain GLP-1 compositions
include, but
are not limited to, insulin releasing agents, inhibitors of glucagon
secretion, protease
inhibitors, glucagon antagonists, anti-obesity agents, compounds that reduce
caloric
intake, selective estrogen receptor modulators, steroid or non-steroid
hormones, growth
factors, and dietary nutrients.
Such additional therapeutic agents can include, for instance, agents for
treating
hyperglycemia, diabetes, hypertension, obesity and bone disorders. Examples of
other
therapeutic agents for treating diabetes that can be included in the
compositions include
those used in treating lipid disorders. Specific examples of such agents
include, but are
not limited to, bile acid sequestrants (e.g., cholestyramine, lipostabil,
tetrahydrolipstatin),
HMG-CoA reductase inhibitors (see, e.g., U.S. Patent Nos. 4,346,227;
5,354,772;
5,177,080; 5,385,929; and 5,753,675), nicotinic acid, MTP inhibitors (see,
e.g., U.S.
Patent Nos. 5,595,872; 5,760,246; 5,885,983; and 5,962,440), lipoxygenase
inhibitors,
fibric acid derivatives, cholesterol absorption inhibitors, squalene
synthetase inhibitors
(see, e.g., U.S. Patent Nos. 4,871,721; 5,712,396; and 4,924,024) and
inhibitors of the
ileal sodium/bile acid cotransporter. Other anti-diabetic agents that can be
incorporated
into the compositions include meglitinides, thiazolidinediones, biguanides,
insulin
secretagogues, insulin sensitizers, glycogen phosphorylase inhibitors, PPAR-
alpha
agonists, PPAR-gamma agonists.
An inhibitor of dipeptidylpeptidase IV activity can also be included to
inhibit
cleavage at the N-terminus of the GLP-1 analog.
The pharmaceutical compositions can include, depending on the formulation
desired, pharmaceutically acceptable, non-toxic carriers or diluents, which
are defined as
vehicles commonly used to formulate pharmaceutical compositions for animal or
human
administration. The diluent is selected so as not to affect the biological
activity of the
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combination. Examples of such diluents are distilled water, buffered water,
physiological
saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. In
addition, the
pharmaceutical composition or formulation can include other carriers,
adjuvants, or non-
toxic, nontherapeutic, nonimmunogenic stabilizers, excipients and the like.
The
compositions can also include additional substances to approximate
physiological
conditions, such as pH adjusting and buffering agents, toxicity adjusting
agents, wetting
agents and detergents.
The composition can also include any of a variety of stabilizing agents, such
as an
antioxidant for example. In cases such as this where the pharmaceutical
composition
includes a polypeptide (e.g., the GLP-1 analog), the polypeptide can be
complexed with
various well-known compounds that enhance the in vivo stability of the
polypeptide, or
otherwise enhance its pharmacological properties (e.g., increase the half-life
of the
polypeptide, reduce its toxicity, enhance solubility or uptake). Examples of
such
modifications or complexing agents include sulfate, gluconate, citrate and
phosphate.
The GLP-1 analog of a composition can also be complexed with molecules that
enhance
their in vivo attributes. Such molecules include, for example, carbohydrates,
polyamines,
amino acids, other peptides, ions (e.g., sodium, potassium, calcium,
magnesium,
manganese), and lipids.
The pharmaceutical compositions can also be formulated as part of a controlled-
release system. Such systems can include an implantable osmotic pump,
liposomes or a
transdermal patch. Methods for delivery using pumps are described, for
example, by
Langer, 1990, Science 249:1527-33; and Saudek et al., 1989, N Engl. J. Med.
321:574).
Delivery options for using liposomes are discussed, for instance, by Treat et
al,, 1989, in
Liposomes in the Terapy of Infetious Disease and Cancer, (Lopez-Berestein and
Fidler,
eds.), Liss, New York, pp. 353-65; and Langer, 1990, Science 249:1527-33).
Further guidance regarding formulations that are suitable for various types of
administration can be found in Remington's Pharmaceutical Sciences, 1985, Mace
Publishing Company, Philadelphia, Pa., 17th ed. For a brief review of methods
for drug
delivery, see, Langer, 1990, Science 249:1527-1533.
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2. Dosage
As noted above, the pharmaceutical compositions can be administered for
prophylactic and/or therapeutic treatments. An "effective amount" refers
generally to an
amount that is a sufficient, but non-toxic, amount of the active ingredient
(i.e., GLP-1
compound or GLP-1 analog) to achieve the desired effect, which is a reduction
or
elimination in the severity and/or frequency of symptoms and/or improvement or
remediation of damage. A "therapeutically effective amount" refers to an
amount that is
sufficient to remedy a disease state or symptoms, or otherwise prevent,
hinder, retard or
reverse the progression of a disease or any other undeirable symptom. A
"prophylactically effective amount" refers to an amount that is effective to
prevent,
hinder, or retard the onset of a disease state or symptom.
In general, toxicity and therapeutic efficacy of the GLP-1 compound can be
determined according to standard pharmaceutical procedures in cell cultures
and/or
experimental animals, including, for example, determining the LD50 (the dose
lethal to
50% of the population) and the ED50 (the dose therapeutically effective in 50%
of the
population). The dose ratio between toxic and therapeutic effects is the
therapeutic index
and it can be expressed as the ratio LD50/ED50. Compositions that exhibit
large
therapeutic indices are desirable.
The data obtained from cell culture and/or animal studies can be used in
formulating a range of dosages for humans. The dosage of the active ingredient
typically
lines within a range of circulating concentrations that include the ED50 with
little or no
toxicity. The dosage can vary within this range depending upon the dosage form
employed and the route of administration utilized.
The amount of active ingredient administered will depend upon various factors
that can be assessed by the attending clinician, such as the severity of the
disease, the age
and size of the subject to be treated and the particular disease itself. In
general, however,
the total amount of the GLP-1 analog itself that is administered typically
ranges from 1
gig/kg body weight/day to 100 mg/kg/day. In some instances, the dosage ranges
from 10
ug/kg /day to 10 mg/kg/day. In other treatment regimens, the GLP-1 compound is
administered at 50 ug/kg/day to 5 mg/kg/day or from 100 ug/kg/day to 1
mg/kg/day.
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C. Administration
The pharmaceutical compositions described herein can be administered in a
variety of different ways. Examples include administering a composition
containing a
pharmaceutically acceptable carrier via oral, intranasal, rectal, topical,
intraperitoneal,
intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal,
and
intracranial methods.
For oral administration, the active ingredient can be administered in solid
dosage
forms, such as capsules, tablets, and powders, or in liquid dosage forms, such
as elixirs,
syrups, and suspensions. The active component(s) can be encapsulated in
gelatin
capsules together with inactive ingredients and powdered carriers, such as
glucose,
lactose, sucrose, fnannitol, starch, cellulose or cellulose derivatives,
magnesium stearate,
stearic acid, sodium saccharin, talcum, magnesium carbonate. Examples of
additional
inactive ingredients that may be added to provide desirable color, taste,
stability,
buffering capacity, dispersion or other known desirable features are red iron
oxide, silica
gel, sodium lauryl sulfate, titanium dioxide, and edible white ink. Similar
diluents can be
used to make compressed tablets. Both tablets and capsules can be manufactured
as
sustained release products to provide for continuous release of medication
over a period
of hours. Compressed tablets can be sugar coated or film coated to mask any
unpleasant
taste and protect the tablet from the atmosphere, or enteric-coated for
selective
disintegration in the gastrointestinal tract. Liquid dosage forms for oral
administration
can contain coloring and flavoring to increase patient acceptance.
The active ingredient, alone or in combination with other suitable components,
can be made into aerosol formulations (i.e., they can be "nebulized") to be
administered
via inhalation. Aerosol formulations can be placed into pressurized
acceptable
propellants, such as dichlorodifluoromethane, propane, nitrogen.
Suitable formulations for rectal administration include, for example,
suppositories, which consist of the packaged active ingredient with a
suppository base.
Suitable suppository bases include natural or synthetic triglycerides or
paraffin
hydrocarbons. In addition, it is also possible to use gelatin rectal capsules
which consist
of a combination of the packaged active ingredient with a base, including, for
example,
liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.
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Formulations suitable for parenteral administration, such as, for example, by
intraarticular (in the joints), intravenous, intramuscular, intradermal,
intraperitoneal, and
subcutaneous routes, include aqueous and non-aqueous, isotonic sterile
injection
solutions, which can contain antioxidants, buffers, bacteriostats, and solutes
that render
the formulation isotonic with the blood of the intended recipient, and aqueous
and non-
aqueous sterile suspensions that can include suspending agents, solubilizers,
thickening
agents, stabilizers, and preservatives.
The components used to formulate the pharmaceutical compositions are
preferably of high purity and are substantially free of potentially harmful
contaminants
(e.g., at least National Food (NF) grade, generally at least analytical grade,
and more
typically at least pharmaceutical grade). Moreover, compositions intended for
in vivo use
are usually sterile. To the extent that a given compound must be synthesized
prior to use,
the resulting product is typically substantially free of any potentially toxic
agents,
particularly any endotoxins, which may be present during the synthesis or
purification
process. Compositions for parental administration are also sterile,
substantially isotonic
and made under GMP conditions.
EXAMPLES
The following examples, including the experiments conducted and results
achieved are provided for illustrative purposes only and are not to be
construed as
limiting the invention.
Example 1
Synthesis of GLP-1 Compounds
Peptide Synthesis
The following protocol was used to generate GLP-1 analogs as described herein.
1\1'.-Fmoc, side-chain protected amino acids, Wang resin, and Rink amide resin
were
used. The following side-chain protection strategies were employed: Asp(OtBu),
Arg(Pbf), Cys(Acm), Glu(OtBu), Glu(02-PhiPr), His(Trt), Lys(1\r-Boc), Lys(Nc-
Mtt),
Ser(OtBu), Thr(OtBu) and Tyr(OtBu). GLP-1 peptide derivatives were synthesized
in a
stepwise manner on an ABI433 peptide synthesizer by SPPS using O-Benzotriazole-
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N,N,N' ,N' -tetramethyl-uronium-hexafluoro-phosphate (HBTIJ)/
N,N-
diisopropylethylamine (DIEA)/ N,N-dimethylformamide (DMF) coupling chemistry
at
0.2 mmol equivalent resin scale (Fmoc-deprotected Rink amide resin). For each
coupling
cycle, 1 mmol N".-Fmoc-amino acid, 4 mmol DIEA and 1 mmol equivalents of HBTIJ
were used. The concentration of the HBTU-activated Fmoc amino acids was 0.5 M
in
DMF, and the coupling time was 45 min. Fmoc deprotections were carried out
with two
treatments using a 30% piperidine in DMF solution first for 2 min and then for
an
additional 20 min.
Lactam Formation
Side-chain to side-chain lactam formation was carried out on the assembled N-
terminally Fmoc-protected peptide resin. The peptide-resin was solvated in DCM
for 30
mins, and drained. The Mtt and 2-PhiPr groups (protecting at the specified
lactam bond
forming site) were removed with 1% TFA in DCM solution containing 5% TIS.
Treatment of the peptide-resin with the 1% TFA in DCM solution was repeated 8
times
in 30 min increments, and each treatment was followed by extensive DCM washes.
The
liberate carboxyl and amino groups were then condensed by the addition of 5
equiv of
0.5M benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophosphate
(PyBOP) and 10 equiv of DIEA in DMF were added to the peptide resin, and left
for 24h.
The resin was then wash thoroughly with DMF, DCM, and DCM/Me0H, and dried.
Side Chain Deprotection and Cleavage from Resin
Following synthesis and modification, the resin was then drained, and washed
with DCM, DMF, DCM, and then dried in vacuo. The peptide-resin was deprotected
and
released from the resin by treatment with a trifluoroacetic acid (TFA)/1,2-
ethanedithiol
(EDT)/triisopropyl-silane (TIS)/H20 (92.5:2.5:2.5:2.5 v/v) solution at room
temperature
for 90 min. The volatiles were then removed with a stream of nitrogen gas, the
crude
peptide precipitated twice with diethyl ether and collected by centrifugation.
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Acm deprotection
Crude GLP-1 Cys(Acm)-protected peptide was dissolved in 10% aq AcOH
containing freshly added mercury(II) acetate (15 mg/mL). The solution was
agitated at
ambient temperature for 4 hours. 80% aq. 2-mercaptoethanol was then added to a
give
20% v/v composition, mixed thoroughly, and left overnight. It was then diluted
with
0.1% aq. TFA, and the grey precipitate containing mercury salts was removed by
centrifugation and filtration. The deprotected peptide was then subjected to
reversed-
phase HPLC purification.
Specific Examples
A synthetic protocol for synthesizing a GLP-1 compound without a ring is
presented in Figure 3, which illustrates the approach for synthesizing an
analog that
includes a Gly substitution at position 8, an Aib substitution at position 22
and the
addition of a cysteine and an alanine at the C-terminus (i.e., SEQ ID NO:18).
Figures 4-11 illustrate the approach for synthesizing a variety of different
cyclic
GLP-1 compounds. Figure 4, for instance, shows a scheme for forming a cyclic
GI,P-1
compound in which the side chains of a glutamic acid and lysine residue are
joined to
form a cyclic lactam. Figure 5, provides an approach for synthesizing a cyclic
GLP-1
compound in which the side chains of a glutamic acid and ornithine side chain
are joined
to form a cyclic lactam. Figure 6, provides an approach for synthesizing a
cyclic GLP-1
compound in which the side chains of an aspartic acid and lysine side chain
are joined to
form a cyclic lactam. Figure 7, provides an approach for synthesizing a cyclic
GLP-1
compound in which the side chains of an aspartic acid and ornithine side chain
are joined
to form a cyclic lactam. Figure 8, provides an approach for synthesizing a
cyclic GLP-1
compound in which the side chains of a lysine and glutamic acid chain are
joined to form
a cyclic lactam. Figure 9, provides an approach for synthesizing a cyclic GLP-
1
compound in which the side chains of a homoglutamic acid and lysine side chain
are
joined to form a cyclic lactam. Figure 10, provides an approach for
synthesizing a cyclic
GLP-1 compound in which the side chains of a 4-carboxy-phenylalanine and
lysine side
chain are joined to form a cyclic lactam. Figure 11, provides an approach for
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synthesizing a cyclic GLP-1 compound in which the side chains of a beta-
Homoglutamic
acid and lysine side chain are joined to form a cyclic lactam.
Reversed-Phase HPLC Purification
Reversed-phase high-performance liquid chromatography was performed on an
analytical (C18, 5 pm, 0.46 cm x 25 cm) or a preparative (C18, 10 .tm, 2.2 cm
x 25 cm)
column. Chromatographic separations were achieved using linear gradients of
buffer B
in A (A = 0.1% aqueous TFA; B = 90% aq. ACN containing 0.09% TFA) typically 5-
95% over 35 min at a flow rate of 1 mUrnin for analytical analysis and 5-65%
over 90
min at 20 mL/min for preparative separations. Analytical and preparative HPLC
fractions
were characterized by ESMS and photodiode array (PDA) HPLC, and selected
fractions
combined and lyophilized.
Mass Spectrometry
Mass spectra were acquired on a single quadrupole mass spectrometer equipped
with an Ionspray atmospheric pressure ionization source. Samples (25 4) were
injected
into a moving solvent (10 4/min; 30:50:20 ACN/Me0H containing 0.05% TFA)
coupled directly to the ionization source via a fused silica capillary
interface (50 jim i.d.).
Sample droplets were ionized at a positive potential of 5 kV and entered the
analyzer
through an interface plate and subsequently through an orifice (100-120 1.1M
diameter) at
a potential of 60 V. Full scan mass spectra were acquired over the mass range
400-2200
Da with a scan step size of 0.1 Da. Molecular masses were derived from the
observed m/z
values.
Pegylation
The thioether-linked PEG-peptides were derived from GLP analogs with reactive
cysteine thiols engineered at the desired conjugation site (See Table 2). The
activated
PEG derivatives were all mono-functional methoxyPEG-maleimides (mPEG-mal) with
MWs of 5kD to 40kD. Conjugation was achieved by alkylation at pH 6. Briefly,
the
peptide was dissolved at 2 mg/ml in an amine-free buffer (50mM sodium
phosphate,
5mM EDTA, pH 6), the mPEG-mal was added in a modest stoichiometric excess (1.2-
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fold) and allowed to react 0.5-2 hrs at room temperature. The reaction was
monitored by
reverse phase HPLC, quenched with 5mM p-mercaptoethanol, allowed to incubate
at
room temperature another 30 min and then purified.
Purification was achieved by preparative cation-exchange chromatography using
SP Sepharose HP (GE Healthcare) and eluting with a linear 0-500mM sodium
chloride
gradient. The eluted PEG-peptide was evaluated by RP-HPLC and SDS-PAGE, pooled
then concentrated and dialyzed into 10mM sodium acetate, 5% sorbitol, and pH
4.
Purities of >99% were determined for all the final pools by RP-HPLC. Peptide
mapping
and sequencing were used to confirm conjugation with PEG at each of the
targeted
I 0 attachment sites.
Example 2
In vitro assays
In vitro Efficacy of GLP-1 Constructs
A. GLP-1R Reporter Assay:
To compare the potency of test compounds with GLP-1, reporter cell lines
expressing human or mouse GLP-1 receptors were generated. Increased cAMP
levels
were measured through enhanced expression of a luciferase reporter gene.
Briefly,
CHOK1 cells expressing the mouse or human GLP-1 receptor, in addition to
harboring a
luciferase reporter gene construct regulated by cyclic AMP levels, were plated
2 days
prior to the assay, then cultured at 37 C, 5% CO2. The evening prior to assay,
the cells
were washed, the medium replaced with serum-free medium containing 0.5%
protease-
free bovine serum albumin (BSA), and then cultured overnight. Cells were
exposed to a
range of concentrations of test compound or GLP-1 for a period of 6 hours at
37 C in
medium containing 0.5% protease-free BSA and 100 riM IBMX. Cell lysates were
assayed for luciferase activity using the Luciferase Assay System (Promega
Corporation,
Madison, WI). Luciferase activity was measured using a LuminoskanTM Ascent
(Thermo
Electron Corporation, Marietta, OH). Nonlinear regression analyses of
resultant
* Trade-mark
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compound concentration curves were performed using GraphPad P
(GraphPad
Software, Inc., San Diego, CA). The "EC50" represents the concentration of
compound at
which 50 percent of the maximal activity is achieved.
B. In vitro GLP-1 Receptor Binding of Constructs
Membrane Preparation. CHOK1 cells expressing either human mouse GLP-1
receptor were harvested from 150 mm culture dishes using PBS. Cells were
sedimented
at 1500 rpm for 10 minutes. The resulting pellets were homogenized in 15 mls
of ice
cold sucrose buffer (25 mM Tris-1-IC1, 0.32 M Sucrose, 0.25 g/L sodium azide,
pH 7.4)
with a motorized, glass fitted, Teflon*homogenizer. The homogenate was
centrifuged at
48,000 X g at 4 C for 10 minutes, resuspended in 25 ml assay buffer (50 mM
Tris-HC1,
TM
5 mM MgC12, 10 mg/ml protease-free BSA, 0.1 mg/ml STI, and 0.1 mg/ml Pefabloc,
pH
7.4) with a Tissue-Tearor (Biospec Products), then centrifuged again at 48,000
X g for 10
minutes. The pellets were homogenized for a third time in 15 ml assay buffer
using the
Tissue-Tearor and again centrifuged at 48,000 X g for 10 minutes. The
resulting pellet
was resuspended in assay buffer at a wet weight concentration of 4 mg/ml.
Ligand Binding Assay. Binding assays were performed in 96-well U-bottom
plates. Membranes (200 1.Lg tissue) were incubated at room temperature for 2
hours in
assay buffer containing 0.2 nM 125I-GLP-1 (PerkinElmer Life Sciences, Boston,
MA) and
with a range of concentrations of test compound or GLP-1 in a total volume of
100 In
addition, non-specific binding was assessed in the presence of 1 t11/1
unlabeled GLP-1.
The reaction was terminated by rapid filtration through Unfilter-96 GF/C glass
fiber filter
plates (FilterMatem196 Packard Harvester, PerkinElmer, Shelton, CT) pre-soaked
in 0.5%
polyethylenimine, followed by three washes with 300 p.1 of cold 50 mM Tris-
HCI, pH
7.4. Bound radioactivity was determined using a TopCount microplate
scintillation and
luminescence counter (Packard Instrument Company, PerkinElmer, Shelton, CT).
Nonlinear regression analyses of resulting concentration curves were performed
using
GraphPad Prism (GraphPad Software, Inc., San Diego, CA). The "IC50" represents
the
* Trade-mark
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concentration of compound that reduces the maximal specific 1251-GLP-1 binding
by 50
percent.
Example 3
In vivo Assays
A. Db/db mice:
The db/db diabetic mouse model was used in this screen to further examine GLP-
1 compounds in regard to fed blood glucose, with this measurement monitored at
1, 2, 4,
6 and 24h. The db/db mice are commercially available from The Jackson
Laboratory
JAXO GEMMO Strain - Spontaneous Mutation Congenic Mice, and are homozygous for
the diabetes spontaneous mutation (Leprdb). These mice become identifiably
obese
around 3 to 4 weeks of age. The criterion for selection for each mouse to
enter the study
was blood glucose of at least 300 mg/dL. Db/db mice at 8.5 weeks of age (for a
chronic
1-2wk study) to about 10-11weeks of age (for an acute 1-3 day study) were
injected once
with each tested compound (acute experiment) or multiple times (chronic
experiment).
On the day of the experiment, the mice were bled at 9 am (baseline value) and
then
immediately handed over to the injector, who then injected the appropriate GLP-
1
compound or +/- control. The mice were then placed in a fresh cage without any
chow, so
as to limit any variability in blood glucose levels associated with eating
behaviors. Time
points of 1 hr, 4hr, 6hr, and 24hr were normally taken. When at the 24hour
time point
blood glucose values were below where they started, further time points at
every 24hrs
were taken until blood glucose returned to the baseline levels. Normal chow
was given
back after the 6hr time point.
Tachyphylaxis was determined by multiple injections. A second injection of the
compound was administered after the blood glucose levels sufficiently returned
to
starting baseline levels. At this point, it was evident whether the compound
had the same
effect/efficacy, or if there was any noticeable tachyphylaxis.
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B. C57b16 mice:
C57B16 (normal lean) mice were used at 10 to 12 weeks of age. These mice are
commercially available through any vendor, such as Jackson Laboratories or
Charles
River, and are considered to be normal. The term "lean" is used to contrast
these mice to
obese db/db mice. C57B16 mice were randomized on body weight. 9 am bleed was
performed to determine baseline blood glucose and GLP-1 compounds or PBS was
administered prior to place the mice in a cage without food. After 4-5hrs, an
intraperitoneal glucose tolerance test (glucose tolerance test measures the
body's ability
to metabolize glucose) was performed using 2g/kg of glucose dose. Blood
glucose levels
were measured 30 min and 90 minutes after the glucose load was administered
and 24
hours or until blood glucose levels were back to the original values. From
these studies,
the enhanced effect of GLP-1 action in utilizing glucose can be seen as
opposed to (-)
control PBS.
Example 4
Results with GLP-1 Compounds Related to Formula I
In vitro experiments were conducted to determine receptor binding and potency
for a class of molecules having a glycine substitution at position 8, a
cysteine substitution
at position 22 and a variable C-terminus. Experiments were performed as
described in
Example 2, and the results are summarized in Table 3 below.
Table 3
INTERNAL REF SEQUENCE IC so EC50
EC50
human human
MOUOG
HGEGT FTSDV SSYLE GCAAK EFIAW LVKGR G (SEQ ID NO: 0.24
1.4
, 6) 70 nM nM nM
cgGLP-1 245
SEQ ID NO: 6 + 20K PEG >1 uM 12 nM
nM
cgGLP-2 3.6
SEQ ID NO: 6 + 40k branched PEG >1 uM 0.7 nM nM
cgGLP-3 5.7
SEQ ID NO: 6 + 8k PEG dumbbell 84 nM 0.2 nM nM
c9GLP-7 2.9
SEQ ID NO: 6 + 30k PEG >1 uM 0.2 nM nM
cgGLP-9 SEQ ID NO: 6 + 20k PEG dimer >1 uM 1 nM 9
nM
HGEGT FTSDV SSYLE GCAAK EFIAW LVKGR GSSGA PPPS 100 0.8
(SEQ ID NO: 7) nM 0.1 nM nM
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cgGLP-12 SEQ ID NO: 7 + 20k PEG monomer 1 uM 1 nM 8
nM
cgGLP-14 300
SEQ ID NO: 7 + 20k PEG dimer nM 0.6 nM 4
nM
HGEGT FTSDV SSYLE GCAAK EFIAW LKNGG PSSGA PPPS 100 0.07 0.3
(SEQ ID NO: 8) nM nM ON
c5GLP-13 SEQ ID NO: 8 + 20k PEG monomer >1 uM 1 nM 3
nM
cgGLP-15 300
SEQ ID NO: 8 + 20k PEG dimer nM 0.8 nM 2
nM
As indicated in Table 3, some of these molecules were conjugated to different
forms of PEG, with the conjugation occurring at the cysteine at position 23.
For instance,
a 20k PEG dimer is a single 20 kD linear PEG polymer with two peptides, both
attached
at the same end like a fork. "Forked" PEG-(maleimide)2 can be obtained from
Nektar
Therapeutics (Huntsville, AL, cat. #2D2M0P0F) and has the following structure:
Catalog Number: 21)2DOPOF
Product Name: M-PEG-(MAI,),, AINV 20,000
Lot Number: PT-02D-17
.
NHCH2CII2NHCOCH2CH2N..,
0 0 f¨CII
0
CI-13-0-PEG-0-C112¨CH2-CI ¨NH
A
CH
0
NIICII2CH2NIICOCH2C112N:, II
1
()a
"Branched" PEG conjugates are 2 polymers attached at the same site on a single
peptide. Branched PEG2-maleimide can also be obtained from Nektar Thearpeutics
(Huntsville, AL), under cat# 2D3X0P11 and has the following structure:
Catalog Number: 2D3X0P11
Product Name: mPEG2-MAL, MW 20,000
Lot Number: PT-02B-15
cH3o(cH2cH2o)4m-i
(1-12)4
0 CH
CH30(CH2CH20)nONH CNH(CH2)2NHC(CH2)2N).)
8
Other molecules (e.g., cgGLP-3) is a "dumbbell" PEG conjugate. In this
configuration 2 peptides are conjugated to an 8kD PEG, one at each end, like a
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"dumbbell". The 8kD mal-PEG-mal polymer is also available from Nektar
Therapeutics
(Huntsville, AL), cat#ZF-066-05 and has the following structure:
0
9 9
`NcH2cH,0\TH(cH,,cw,0),,cHcH,Nficaf,cH,N,
0 0
Example 5
Results with GLP-1 Compounds Related to Formula II
In vitro experiments were conducted to determine receptor binding and potency
for GLP-1 compounds having a substitution at position 8 and either a CSG or
CSGG C-
termnial addition. The pegylated molecules were conjugated at the cysteine at
position
38. Experiments were conducted as described in Example 2, and the results are
summarized in Table 4 below.
Table 4
INTERNAL REF # SEQUENCE IC50 EC,, EC,,
human human mouse,
HGEGT FTSDV SSYLE GQAAK EFIAW LVKGR GCSG
(SEQ ID NO: 10) 17 nM
0.05 nM 0.32 nM
cgGLP-4 & mgGLP-09A SEQ ID NO: 10 + 20k PEG
130 nM 0.23 nM 1.8 nM
cgGLP-5 SEQ ID NO: 10 40k branched PEG
>1 uM 0.3 nM 1.5 nM
cgGLP-6 SEQ ID NO: 10 + 8k PEG dumbbell 11 nM -
0.1 nM 0.8 nM
cgGLP-8 SEQ ID NO: 10 + 30k PEG 170 nM 0.4 nM
3 nM
cgGLP-10 SEQ ID NO: 10 + 20k PEG dimer 100 nM 0.6 nM
4 nM
cgGLP-16 SEQ ID NO: 10 + 30k PEG I uM 0.39 nM
4 nM
H[Aib]EGT FTSDV SSYLE GQAAK EFIAW LVKGR
GCSGG (SEQ ID NO: 11) 100 nM 4
nM >50 nM
cgGLP-17
SEQ ID NO: 11 + 20k PEG 1 uM 20 nM
>100 nM
H[bAla]EGT FTSDV SSYLE GQAAK EFIAW LVKGR
GCSGG (SEQ ID NO: 12)
400 nM >100 nM undet'd
cgGLP-18
SEQ ID NO: 12 + 20k PEG >1
uM undet'd undet'd
Aib = alpha
aminobutyric acid
bAla = beta
aminopropionic acid
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Example 6
Results with Compounds Related to Formula III
In vitro experiments were conducted to determine receptor binding and potency
for GL,P-1 compounds having either an Aib (2-aminoisobutyric acid) or Aad (2-
aminoadipic acid) substitution at position 22 and optional substitution at
position 8 and at
the C-terminus. The pegylated molecules were conjugated at the cysteine at
position 38.
Experiments were performed as described in Example 2, and the results are
summarized
in Table 5 below.
Table 5
INTERNAL REF # SEQUENCE IC50 EC50 ECso
human human mouse
HGEGT FTSDV SSYLE [Aib)QAAK EFIAW LVKGR - 2.5 0.15
AMIDE (SEQ ID NO: 14) nM 16 pM nM
HGEGT FTSDV SSYLE [Aib)QAAK EFIAW LVKGR G- 3.5 0.12
AMIDE (SEQ ID NO: 15) nM 27 pM nM
cgGLP-23 HGEGT FTSDV SSYLE [AiblQAAK EFIAW LVKGR GC-
AMIDE 300
SEQ ID NO: 17 + 20k PEG nM 0.2 nM 3
nM
HGEGT FTSDV SSYLE [AiblQAAK EFIAW LVKGR GCA-
AMIDE (SEQ ID NO: 18) 25 pM
0.2 nM
mgGLP32 & mgGLP- 0.13 0.84
SEQ ID NO: 18 + 20k PEG 56 nM nM nM
HGEGT FTSDV SSYLE [Aib]QAAK EFIAW LVKGR GCG-
AMIDE (SEQ ID NO: 20)
mgGLP-16
SEQ ID NO: 20 + 20k PEG 50 nM 0.2 nM 1.6 nM
HGEGT FTSDV SSYLE [Aib]QAAK EFIAW LVKGR GCSG
(SEQ ID NO: 21) 18 nM 68 pM
300 pM
mgGLP19 & cgGLP- 230 0.32
19 SEQ ID NO: 21 + 20k PEG nM nM 4 nM
HGEGT FTSDV SSYLE [AiblQAAK EFIAW LVKGR GCSG- 0,27
AMIDE (SEQ ID NO: 22) 17 nM 45 pM nM
cgGLP-24 cgGLP-
27, mgGLP-22,
m5GLP-33 & mgGLP- 0,84
27 SEQ ID NO: 22 + 20k PEG 47 nM 0.1 nM nM
cgGLP-25 SEQ ID NO: 22 + 5k PEG 15 nM 39 pM
0.9 nM
cgGLP-26 , SEQ ID NO: 22 + 10k PEG 23 nM 59 pM
0.9 nM
cgGLP-28 SEQ ID NO: 22 + 30k PEG 71 nM 0.1 nM 0.8 nM
cgGLP-29 800 0.15
SEQ ID NO: 22 + 40k branched PEG nM nM 1 nM
HGEGT FTSDV SSYLE [Aib]QAAK EFIAW LVKGR
GCSGG- AMIDE (SEQ ID NO: 24)
mgGLP-15 100
SEQ ID NO: 24 + 20k PEG nM
0.2 nM 1.6 nM
H[Aib]EGT FTSDV SSYLE [Aib]QAAK EFIAW LVKGR
GCSG (SEQ ID NO: 25)
cgGLP-20
SEQ ID NO: 25 + 20k mPEG 30 nM 99 pM
1.5 nM
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Example 7
Results with Cyclic GLP-1 Compounds
In vitro experiments were conducted to determine receptor binding and potency
for a variety of GLP-1 compounds in which the side chains of certain amino
acids were
joined to form a ring (a cyclic lactam). Experiments were performed as
described in
Example 2. The sequences of the cyclic compounds tested are shown in Table 6
together
with the results. The side chains of the amino acids involved in forming the
cyclic lactam
are shown in bold type and underlined. Of the compounds listed in Table 6,
only
mgGLP-24 was pegylated.
Table 6
INTERNAL REF # SEQUENCE IC50 EC50 EC50
human human mouse
[G1y8)GLP1(7-37) HGEGT FTSDV SSYEE GQKAK EFIAW LVKGR G
cyclo[G1u14-Ly518] AMIDE (SEQ ID NO: 35) 12 pM
0.21 nM
(01y8)GLP1(7-37) HGEGT FTSDV SSYLE GQAKK EFIAW LVKGR G
_ _
cyc[G1u15-Lys19) AMIDE (SEQ ID NO: 36) 0.15 nM 66
pM
GLP1(7-37)
cyclo[G1u15-Lys19; HGEGT FTSDV SSYLE GQAKK EFIAW LEKGR K
27-31) AMIDE (SEQ ID NO: 277)-
undet'd undet'd
[G1y8]cyclo[21- HGEGT FTSDV SSYLE GQAAK EFIAW LVKGR G
26)GLP1(7-37)-amide AMIDE (SEQ ID NO:42)
0.6 nM undet'd
HGEGT FTSDV SSYLE EQAAK EFIAW LVKGR G - 1.1
cycloE22-K26 AMIDE (SEQ ID NO: -3-0) oN 9 pM 68
pM
mgGLP24
SEQ ID NO: 30 + 20k mPEG 48
nM 0.25 nM 0.9 nM
HGEGT FTSDV SSYLE EQAAO EFIAW LVKGR G - 3.7
cycloE22-026 AMIDE (SEQ ID NO: -6-9) nM 51 pM
0.25 nM
GLP1(7-37) HGEGT FTSDV SSYLE KQAAE EFIAW LVKGR G
cyclo[Lys16-G1u20] AMIDE (SEQ ID NO: 159) 16 pM
0.1 nM
[G1y8]GLP1(7-37) HGEGT FTSDV SSYLE GEAAK KFIAW LVKGR G
cyclo[G1u17-Lys21] _AMIDE (SEQ ID NO: 3-8) 18 pM
0.17 nM
[G1y8)GLP1(7-37) HGEGT FTSDV SSYLE GQEAK EKIAW LVKGR G
cyclo[G1u18-Lys22] AMIDE (SEQ ID NO: 39)-
0.8 nM undet'd
0= ornithine = aa
w/ CH2CH2CH2NH3
side chain
Example 8
Results with GLP-1 Compounds Haying N-terminal Extension
In vitro experiments were conducted to determine receptor binding and potency
for GLP-1 compounds having an N-terminal extension. Experiments were performed
as
described in Example 2, and the results are summarized in Table 7 below. None
of the
compounds listed in Table 7 were pegyalted.
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Table 7
INTERNAL SEQUENCE ICso EC50 EC50
REF # human human
mouse
0.2 0.8
A-GLP-1 A HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR (SEQ ID NO: 247) 38 nM
nM nM
G-GLP-1 G HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR (SEQ ID NO: 248) BO nM
1 nM 3 nM
0.2
P-GLP-1 P HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR (SEQ ID NO: 249) 38 nM
nM 1 nM
0.4
S-GLP-1 .5 HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR (SEQ ID NO: 250) 53 nM
nM 2 nM
0.3
T-GLP-1 T HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR (SEQ ID NO: 251) 53 nM
nM 2 nM
0.2
V-GLP-1 V HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR (SEQ ID NO: 252) 32 nM
nM 1 nM
0.5 3.0
MQ-GLP1 MQ HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR (SEQ ID NO: 253) 64 nM
nM nM
590
MR-GLP1 MR HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR (SEQ ID NO: 254) nM 8
nM 41 nM
760
MK-GLP1 MK HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR (SEQ ID NO: 255) nM 4
nM 12 nM
0.5
M-GLP1 M HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR (SEQ ID NO: 256) 18 nM
86 pM nM
350
MH-GLP1 NH HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR (SEQ ID NO: 257) nM 2
nM 11 nM
MHH HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR (SEQ ID NO: 350 1,0
MHH-GLP1 258) nM nM 5
nM
0.4
MY-GLP1 MY HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR (SEQ ID NO: 259) 57 nM
nM 1 nM
0.6
MI-GLP1 MI HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR (SEQ ID NO: 260) 75 nM
nM 6 nM
0.3
MD-GLP1 MD HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR (SEQ ID NO: 261) 82 nM
nM 2 nM
160 0.7
ML-GLP1 ML HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR (SEQ ID NO: 262) nM
nM 6 nM
0.3
MN-GLP1 MN HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR (SEQ ID NO: 263) 50 nM
nM 2 nM
ME-GLP1 ME HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR (SEQ ID NO: 264) 95 nM
1 nM 4 nM
0.8
MW-GLP1 MW HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR (SEQ ID NO: 265) , 79 nM
nM 5 nM
180
MF-GLP1 MF HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR (SEQ ID NO: 266) nM 1
nM 6 nM
100 0.5
MM-GLP1 MM HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR (SEQ ID NO: 267) nM
nM 3 nM
Example 9
In vivo Results ¨ Effect on Blood Glucose Levels with Time
A variety of different GLP-1 compounds, including representatives from the
different classes disclosed herein (e.g., compounds having the general
structure of
formulas I-TV described above), were tested for their ability to affect blood
glucose
levels. In these experiments, blood glucose was measured in db/db mice as
described in
Example 3.
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Figure 12 shows the ability of cgGLP-17, cgGLP-18, cgGLP-19, mgGLP-09A
(see Tables 4 and 5 for sequences and size and type of PEG) and mg-GLP-09A to
lower
blood glucose over a 72 hour time span. Each of these compounds lowered blood
glucose relative to control, with cg-GLP-19 lowering blood glucose for the
longest period
of time (72 hours was needed at a 10 ug/mouse dose for glucose levels in the
mouse to
return to normal levels). Another set of experiments were conducted with cgGLP-
19,
cgGLP-20, cgGLP-23, cgGLP-24 (see Table 5 for sequences and size and type of
PEG).
ug of GLP-1 compound was injected into each mouse. As shown in Figure 13,
these
four GLP-1 compounds showed similar ability to decrease blood glucose levels
over
10 time, each causing a signficant decrease in blood glucose levels over a
48 hour time
period.
Another set of experiments were conducted using mgGLP-20, cgGLP-26,
mgGLP-22, and mgGLP-24 (see Tables 5 and 6 for sequences and size and type of
PEG). Dosage was 10 ug per mouse. As shown in Figure 14, although each
compound
lowered blood glucose levels, they did so differently. In this example, mgGLP-
20 and
mgGLP-24 lowered glucose for the longest period of time. With these two
compounds,
72 hours was needed for blood glucose levels to return to normal levels.
Example 10
Lowering of Blood Glucose in Dose Dependent Fashion
As described in Example 3, db/db mice were injected with mgGLP-32 (see Table
5 for sequence and PEG size) at different doses (5 and 10 jig/mouse). As shown
in Figure
15, mgGLP-32 lowered blood glucose levels for 24 hours in a dose dependent
fashion.
Example 11
Lean GTT Experiments
GLP-1 action on insulin release is greater in presence of glucose than in the
absence of glucose. C57B16 mice have normal blood glucose, it is then
challenging to
determine differences in efficacy between different GLP-1 analogs (window for
efficacy
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is not very broad). In order to determine efficacy between tested GLP-1
analogs, GTT
was performed in C57B16 mice 52 hours post the GLP-1 analogs or PBS injections
as
described in example 3. Figure 16 shows mgGLP-20, mgGLP-22 and mgGLP-24
decreased blood glucose to the same extent during the first 52 hours after
injection of the
GLP-1 analogs or PBS (see Tables 5 and 6 for sequences and size and type of
PEG
molecule). At 30 min into the OTT, the separation between glucose levels of
the vehicle
treated mice and the GLP-1 analogs treated mice, demonstrating the expected
effect of
GLP-1 analogs on reducing blood glucose. Also, at 30 min mgGLP-24 showed a
greater
efficacy than mgGLP-22 and mgOLP-22 showed a greater efficacy than mgGLP-20 at
lowering blood glucose duringthe GTT.
Example 12
Multi Dose Experiments
To test whether tachyphylaxis was an issue with long acting forms of GLP-1
compounds, a multiple dose experiment was performed in db/db mice. The method
described in Example 3 was used, except that a second bolus of the GLP-1
compound
was injected the next day, right after the 24 hour blood glucose measurement.
As shown
in Figure 17, blood glucose was decreased to the same extent on the second day
with
mgGLP-33 (see Table 5 for sequence and PEG size). No tachyphylaxyis was
observed
with the tested GLP-1 compound. The same result was observed with mgOLP-20
(see
Table 5 for sequence and size and type of PEG).
In addition, a 4-day, multiple dose experiment with mgGLP-32 was performed
(see Table 5 for sequence and size and type of PEG) in normal C57B16 mice.
Also, to
determine efficacy, a GTT was performed after the first injection, and a
second OTT was
performed after the 4th injection. OTT were performed as described in Example
3.
Results showing GLP-1 analogs efficiency on blood glucose are shown in Figure
18.
As shown in Figure 19, blood glucose was decreased during the 4 hour fast with
mgGLP-20. Also, no blood glucose spike was seen during the first and second
GTT in
the mice treated with mgGLP-20. No tachyphylaxyis was observed after the 4th
dose of
mgGLP-20.
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Example 13
Pegylation Studies
All activated PEG polymers were obtained from Nektar Therapeutics (Huntsville,
AL). A discussion of the different forms of PEG molecules used is provided in
Example
4.
Numerous PEG-maleimide polymers are available ranging in size from 5-40 kD,
and may also contain branched PEG polymers or have multivalent functional
groups.
These polymers were used to determine an optimal balance of PEG size, polymer
branching and peptide valence for improved pharmacokinetics with minimal
impact on
receptor binding. GLP-1 analogues were prepared from linear, mono-functional
PEG-
maleimides of: 5 kD, 10 kD, 20 kD and 301(D. Also, a 40 kD branched PEG (2 x
20 kD
polymers) maleimide was tested. The in vitro binding assay highlights an
inverse
relationship between PEG size and receptor affinity, with the branched 40 kD
polymer
having a very significant impact on receptor binding (Tables 3-5). Similarly,
PEG size
influences the duration of glucose decrease in vivo, with larger polymers
typically
achieving the greatest duration (data not shown). However, there is a limit to
this effect
and the largest PEG-GLP analogue (cgGLP-29) with a branched 40 kD polymer was
not
the most active in vivo.
Figures 20 & 21 show results for a GLP-1 reporter assay for the activation of
the
human GLP-1 receptor by SEQ ID NOS: 6 and 10 respectively conjugated to: 1)
two 8
kD PEG polymers, one at each end, like a "dumbbell"; 2) 20 kD PEG polymer; 3)
30 kD
PEG polymer; or 4) 40 kD branched PEG polymer. Interestingly, while SEQ ID
NO:10
shows similar functional activity for the 20, 30 and 40 kD PEG conjugates, SEQ
ID NO:6
shows a marked reduction in activity with the 20 kD conjugate, while the 40 kD
branched
conjugate shows similar potency to the 30 kD and 8 kD dumbbell conjugates.
This is in
contrast to the observed inverse relationship between receptor binding
affinity and PEG
size. Figure 22 shows similar results from the GLP-1 receptor functional assay
using
SEQ ID NO:22 conjugated to: 1) 5 kD PEG polymer; 2) 10 kD PEG polymer; 3) 20
kD
PEG polymer; 4) 30 kD PEG polymer; or 5) 40 kD branched PEG polymer. The EC50
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72249-199
values corresponding to these results are summarized in Tables 3-5. With these
peptide
conjugates, we see similar in vitro potency with all sizes of PEG conjugates
with the
exception of the 5 kD PEG conjugate having potency similar to the unconjugated
peptide.
An in vivo set of experiments were conducted using cgGLP-25 (5 kDa PEG), cgGLP-
26
(10 k Da PEG), cgGLP-27 (20 kDa PEG) and cgGLP-28 (branched 40 kDa PEG) to
determine the effect of different sizes and shapes of PEG have on the ability
to lower
glucose levels. Each of these compounds has the amino acid sequence of SEQ ID
NO:22
but differ in the size or shape of the PEG that is attached as indicated.
Blood glucose was
measured in db/db mice as described in Example 3. The different compounds
lowered
blood glucose differently, with cgGLP-27 lowering blood glucose for the
longest period
of time (Figure 23).
******
It is understood that the examples and embodiments described herein are for
illustrative purposes only and that various modifications or changes in light
thereof will
be suggested to persons skilled in the art and are to be included within the
spirit and
purview of this application and scope of the appended claims.
83
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SEQUENCE LISTING IN ELECTRONIC FORM
In accordance with Section 111(1) of the Patent Rules, this
description contains a sequence listing in electronic form in ASCII
text format (file: 72249-199 Seq 14-MAR-13 v3.txt).
A copy of the sequence listing in electronic form is available from
the Canadian Intellectual Property Office.
The sequences in the sequence listing in electronic form are
reproduced in the following table.
SEQUENCE TABLE
<110> Gegg, Colin
Miranda, Leslie
Winters, Katherine
Veniant-Ellison, Murielle
<120> GLP-1 Compounds
<130> 72249-199
<140> 2,648,936
<141> 2007-04-20
<150> 60/793,707
<151> 2006-04-20
<160> 304
<170> PatentIn version 3.3
<210> 1
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic
<400> 1
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 2
<211> 31
<212> PRT
<213> Artificial Sequence
84
CA 02648936 2013-03-20
<220>
<223> Synthetic
<400> 2
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 3
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 3
Met His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
1 5 10 15
Gly Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
<210> 4
<211> 36
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 4
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Gly
20 25 30
Gly Gly Gly Cys
<210> 5
<211> 39
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC_FEATURE
<222> (1)..(1)
<223> Xaa is: L-histidine, D-histidine, desamino-histidine,
2-amino-histidine, 3-hydroxy- histidine, homohistidine,
a-fluoromethyl-histidine or a-methyl-histidine
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (3)..(3)
<223> Xaa at position 3 is Glu, Asp, or Lys
<220>
<221> MISC FEATURE
<222> (4)..(4)
<223> Xaa at position 4 is Gly or His
<220>
<221> MISC FEATURE
<222> (5)..(5)
<223> Xaa at position 5 is Thr, Ala, Gly, Ser, Leu, Ile, Val, Glu, Asp,
or Lys
<220>
<221> MISC FEATURE
<222> (6)..(6)
<223> Xaa at position 6 is: His, Trp, Phe, or Tyr
<220>
<221> MISC FEATURE
<222> (7)..(7)
<223> Xaa at position 7 is Thr or Gly
<220>
<221> MISC FEATURE
<222> (8)..(8)
<223> Xaa at position 8 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp,
or Lys
<220>
<221> MISC FEATURE
<222> (9)..(9)
<223> Xaa at position 9 is Asp or Glu
<220>
<221> MISC FEATURE
<222> (10)..(10)
<223> Xaa at position 10 is Val, Ala, Gly, Ser, Thr, Leu, Ile, Tyr,
Glu, Asp, Trp, or Lys
<220>
<221> MISC FEATURE
<222> (11)..(11)
<223> Xaa at position 11 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu,
Asp, or Lys
<220>
<221> MISC FEATURE
<222> (12)..(12)
<223> Xaa at position 12 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu,
Asp, Trp, Tyr, Lys, Homolysine, Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid, or homoglutamic acid
86
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (13)..(13)
<223> Xaa at position 13 is Tyr, Phe, Trp, Glu, Asp, Gin, Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-
Homoglutamic acid, or
homoglutamic acid
<220>
<221> MISC FEATURE
<222> (14)..(14)
<223> Xaa at position 14 is Leu, Ala, Gly, Ser, Thr, Ile, Val, Glu,
Asp, Met, Trp, Tyr, Lys, Homolysine, Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (15)..(15)
<223> Xaa at position 15 is Glu, Asp, Lys, Homolysine, Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or
homoglutamic acid
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa at position 16 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu,
Asp, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine,
beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (18)7.(18)
<223> Xaa at position 18 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu,
Asp, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine,
beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (19)..(19)
<223> Xaa at position 19 is Lys, Homolysine, Arg, Gin, Glu, Asp, His,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, or homoglutamic
acid
<220>
<221> MISC _FEATURE
<222> (20)..(20)
<223> Xaa at position 20 is Leu, Glu, Asp, Lys, Homolysine, Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (21)..(21)
<223> Xaa at position 21 is Phe, Trp, Asp, Glu, Lys, Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid,
beta-Homoglutamic acid, or homoglutamic acid
87
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (22)..(22)
<223> Xaa at position 22 is Ile, Leu, Val, Ala, Phe, Asp, Glu, Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic
acid, beta-Homoglutamic acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (23)..(23)
<223> Xaa at position 23 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu,
Asp, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic
acid,
beta-Homoglutamic acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (24)..(24)
<223> Xaa at position 24 is Trp, Phe, Tyr, Glu, Asp, or Lys
<220>
<221> MISC FEATURE
<222> (25)..(25)
<223> Xaa at position 25 is Leu, Gly, Ala, Ser, Thr, Ile, Val, Glu,
Asp, or Lys
<220>
<221> MISC FEATURE
<222> (26)..(26)
<223> Xaa is: Val or Lys
<220>
<221> MISC FEATURE
<222> (27)..(27)
<223> Xaa is: Lys or Asn
<220>
<221> MISC FEATURE
<222> (28)..(28)
<223> Xaa is: Phe, Trp, Asp, Glu, Lys, Homolysine, Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid, or homoglutamic acid;
<220>
<221> MISC FEATURE
<222> (30)..(30)
<223> Xaa is: Gly or Pro
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa at position 31 is Trp, Phe, Tyr, Glu, Asp, or Lys
<220>
<221> MISC FEATURE
88
CA 02648936 2013-03-20
<222> (39)..(39)
<223> Residue 39 is attached to an carboxy group which is attached to
an R1 group, wherein, R1 is 0R2 or NR2R3; R2 and R3 are
independently hydrogen or (C1-C8)alkyl
<400> 5
Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10 15
Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
<210> 6
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 6
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Cys Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 7
<211> 39
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 7
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Cys Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
<210> 8
<211> 39
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 8
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15 .
89
CA 02648936 2013-03-20
Cys Ala Ala Lys Glu Phe Ile Ala Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
<210> 9
<211> 35
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (1)..(1)
<223> Xaa at position 1 is: L-histidine, D-histidine,
desamino-histidine, 2-amino-histidine, 3-hydroxy-histidine,
homohistidine, a-fluoromethyl-histidine or a-methyl-histidine
<220>
<221> MISC FEATURE
<222> (2).7(2)
<223> Xaa at position 2 is Gly, bAla (2-aminopropionic acid),
1-amino-cylcopentanecarboxylic acid, Aib (2-aminoisobutryic acid)
or an alpha-alpha-disubstituted amino acid
<220>
<221> MISC FEATURE
<222> (3)..(3)
<223> Xaa at position 3 is Glu, Asp, or Lys
<220>
<221> MISC FEATURE
<222> (4)..(4)
<223> Xaa at position 4 is Gly or His
<220>
<221> MISC FEATURE
<222> (5)..(5)
<223> Xaa at position 5 is Thr, Ala, Gly, Ser, Leu, Ile, Val, Glu, Asp,
or Lys
<220>
<221> MISC FEATURE
<222> (6)..(6)
<223> Xaa at position 6 is: His, Trp, Phe, or Tyr
<220>
<221> MISC FEATURE
<222> (7)..(7)
<223> Xaa at position 7 is Thr or Gly
<220>
<221> MISC FEATURE
CA 02648936 2013-03-20
<222> (8)..(8)
<223> Xaa at position 8 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp,
or Lys
<220>
<221> MISC FEATURE
<222> (9)..(9)
<223> Xaa at position 9 is Asp or Glu
<220>
<221> MISC FEATURE
<222> (10)..(10)
<223> Xaa at position 10 is Val, Ala, Gly, Ser, Thr, Leu, Ile, Tyr,
Glu, Asp, Trp, or Lys
<220>
<221> MISC FEATURE
<222> (11)..(11)
<223> Xaa at position 11 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu,
Asp, or Lys
<220>
<221> MISC FEATURE
<222> (12)7.(12)
<223> Xaa at position 12 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu,
Asp, Trp, Tyr, Lys, Homolysine, Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (13)..(13)
<223> Xaa at position 13 is Tyr, Phe, Trp, Glu, Asp, Gin, Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic
acid, beta-Homoglutamic acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (14)..(14)
<223> Xaa at position 14 is Leu, Ala, Gly, Ser, Thr, Ile, Val, Glu,
Asp, Met, Trp, Tyr, Lys, Homolysine, Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (15)..(15)
<223> Xaa at position 15 is Glu, Asp, Lys, Homolysine, Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa at position 16 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu,
Asp, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine,
beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid
91
CA 02648936 2013-03-20
=
<220>
<221> MISC FEATURE
<222> (17)..(17)
<223> Xaa at position 17 is Gin, Asn, Arg, Glu, Asp, Lys, Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (18)..(18)
<223> Xaa at position 18 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Arg,
Glu, Asp, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine,
beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (19)..(19)
<223> Xaa at position 19 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu,
Asp, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine,
beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa at position 20 is Lys, Homolysine, Arg, Gin, Glu, Asp, His,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, or
homoglutamic acid
<220>
<221> MISC FEATURE
<222> (21)..(21)
<223> Xaa at position 21 is Leu, Glu, Asp, Lys, Homolysine, Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (22)..(22)
<223> Xaa at position 22 is Phe, Trp, Asp, Glu, Lys, Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid,
beta-Homoglutamic acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (23)..(23)
<223> Xaa at position 23 is Ile, Leu, Val, Ala, Phe, Asp, Glu, Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic
acid, beta-Homoglutamic acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (24)..(24)
<223> Xaa at position 24 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu,
Asp, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine,
beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid
92
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (25)..(25)
<223> Xaa at position 25 is Trp, Phe, Tyr, Glu, Asp, or Lys
<220>
<221> MISC FEATURE
<222> (26)..(26)
<223> Xaa at position 26 is Leu, Gly, Ala, Ser, Thr, Ile, Val, Glu,
Asp, or Lys
<220>
<221> MISC FEATURE
<222> (27)..(27)
<223> Xaa at position 27 is Val, Gly, Ala, Ser, Thr, Leu, Ile, Glu,
Asp, or Lys
<220>
<221> MISC FEATURE
<222> (28)..(28)
<223> Xaa at position 28 is Asn, Lys, Arg, Glu, Asp, or His
<220>
<221> MISC FEATURE
<222> (29)..(29)
<223> Xaa at position 29 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu,
Asp, or Lys
<220>
<221> MISC FEATURE
<222> (30)..(30)
<223> Xaa at position 30 is Gly, Arg, Lys, Glu, Asp, or His
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa at position 31 is Pro, Gly, Ala, Ser, Thr, Leu, Ile, Val,
Glu, Asp, or Lys
<220>
<221> MISC_FEATURE
<222> (35)..(35)
<223> Residue 35 is attached to an carboxy group which is attached to
an R1 group, wherein, R1 is 0R2 or NR2R3; R2 and R3 are
independently hydrogen or (C1-C8)alkyl
<400> 9
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys
20 25 30
Ser Gly Gly
<210> 10
<211> 34
93
CA 02648936 2013-03-20
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 10
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ser Gly
<210> 11
<211> 35
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (2)..(2)
<223> Xaa is Aib
<400> 11
His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ser Gly Gly
<210> 12
<211> 35
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (2)..(2)
<223> Xaa- is bAla
<400> 12
His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ser Gly Gly
94
CA 02648936 2013-03-20
<210> 13
<211> 35
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (1)..(1)
<223> Xaa at position 1 is: L-histidine, D-histidine,
desamino-histidine, 2-amino-histidine, 3-hydroxy-histidine,
homohistidine, a-fluoromethyl-histidine or a-methyl-histidine
<220>
<221> MISC FEATURE
<222> (2)..(2)
<223> Xaa at position 2 is Gly, bAla (2-aminopropionic acid),
1-amino-cylcopentanecarboxylic acid, Aib (2-aminoisobutryic acid)
or an alpha-alpha-disubstituted amino acid
<220>
<221> MISC FEATURE
<222> (3)..(3)
<223> Xaa at position 3 is Glu, Asp, or Lys
<220>
<221> MISC FEATURE
<222> (4)..(4)
<223> Xaa at position 4 is Gly or His
<220>
<221> MISC FEATURE
<222> (5)..(5)
<223> Xaa at position 5 is Thr, Ala, Gly, Ser, Leu, Ile, Val, Glu, Asp,
or Lys
<220>
<221> MISC FEATURE
<222> (6)..(6)
<223> Xaa at position 6 is His, Trp, Phe, or Tyr
<220>
<221> MISC FEATURE
<222> (7).7(7)
<223> Xaa at position 7 is Thr or Gly
<220>
<221> MISC FEATURE
<222> (8)..(8)
<223> Xaa at position 8 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp,
or Lys
<220>
<221> MISC FEATURE
CA 02648936 2013-03-20
=
<222> (9)..(9)
<223> Xaa at position 9 is Asp or Glu
<220>
<221> MISC FEATURE
<222> (10)..(10)
<223> Xaa at position 10 is Val, Ala, Gly, Ser, Thr, Leu, Ile, Tyr,
Glu, Asp, Trp, or Lys
<220>
<221> MISC FEATURE
<222> (11)..(11)
<223> Xaa at position 11 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu,
Asp, or Lys
<220>
<221> MISC FEATURE
<222> (12)..(12)
<223> Xaa at position 12 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu,
Asp, Trp, Tyr, Lys, Homolysine, Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (13)..(13)
<223> Xaa at position 13 is Tyr, Phe, Trp, Glu, Asp, Gln, Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic
acid, beta-Homoglutamic acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (14)..(14)
<223> Xaa at position 14 is Leu, Ala, Gly, Ser, Thr, Ile, Val, Glu,
Asp, Met, Trp, Tyr, Lys, Homolysine, Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (15)..(15)
<223> Xaa at position 15 is Glu, Asp, Lys, Homolysine, Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (16)7.(16)
<223> Xaa at position 16 is Aib (2-aminoisobutyric acid),
1-amino-cylcopentanecarboxylic acid, an alpha-alpha-disubstituted
amino acid, or Aad (2-aminoadipic acid)
<220>
<221> MISC FEATURE
<222> (17)..(17)
<223> Xaa at position 17 is Gln, Asn, Arg, Glu, Asp, Lys, Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid, or homoglutamic acid
96
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (18)..(18)
<223> Xaa at position 18 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Arg,
Glu, Asp, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine,
beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (19)..(19)
<223> Xaa at position 19 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu,
Asp, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine,
beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa at position 20 is Lys, Homolysine, Arg, Gin, Glu, Asp, His,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, or
homoglutamic acid
<220>
<221> MISC FEATURE
<222> (21)..(21)
<223> Xaa at position 21 is Leu, Glu, Asp, Lys, Homolysine, Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (22)..(22)
<223> Xaa at position 22 is Phe, Trp, Asp, Glu, Lys, Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid,
beta-Homoglutamic acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (23)..(23)
<223> Xaa at position 23 is Ile, Leu, Val, Ala, Phe, Asp, Glu, Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic
acid, beta-Homoglutamic acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (24)..(24)
<223> Xaa at position 24 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu,
Asp, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine,
beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (25)..(25)
<223> Xaa at position 25 is Trp, Phe, Tyr, Glu, Asp, or Lys
<220>
<221> MISC FEATURE
97
CA 02648936 2013-03-20
<222> (26)..(26)
<223> Xaa at position 26 is Leu, Gly, Ala, Ser, Thr, Ile, Val, Glu,
Asp, or Lys
<220>
<221> MISC FEATURE
<222> (27)..(27)
<223> Xaa at position 27 is Val, Gly, Ala, Ser, Thr, Leu, Ile, Glu,
Asp, or Lys
<220>
<221> MISC FEATURE
<222> (28)..(28)
<223> Xaa at position 28 is Asn, Lys, Arg, Glu, Asp, or His
<220>
<221> MISC FEATURE
<222> (29)..(29)
<223> Xaa at position 29 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu,
Asp, or Lys
<220>
<221> MISC FEATURE
<222> (30)7.(30)
<223> Xaa at position 30 is Gly, Arg, Lys, Glu, Asp, or His
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa at position 32 is Cys, Gly, or is omitted
<220>
<221> MISC FEATURE
<222> (33)..(33)
<223> Xaa at position 33 is Ala, Gly, Ser, Cys, or is omitted
<220>
<221> MISC FEATURE
<222> (34)..(34)
<223> Xaa at position 34 is Gly or is omitted
<220>
<221> MISC FEATURE
<222> (35)..(35)
<223> Xaa at position 35 is Gly or is omitted
<220>
<221> MISC FEATURE
<222> (35)..(35)
<223> Residue 35 is attached to an carboxy group which is attached to
an R1 group, wherein, R1 is OR2 or NR2R3; R2 and R3 are
independently hydrogen or (C1-C8)alkyl
<400> 13
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10 15
98
CA 02648936 2013-03-20 =
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa
20 25 30
Xaa Xaa Xaa
<210> 14
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is Aib
<220>
<221> MISC_FEATURE
<222> (30)..(30)
<223> residue 30 is amidated
<400> 14
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
<210> 15
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is Aib
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> residue 31 amidated
<400> 15
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
99
CA 02648936 2013-03-20
<210> 16
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is Aib
<400> 16
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
<210> 17
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is Aib
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> residue 32 is amidated
<400> 17
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
<210> 18
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is Aib
100
CA 02648936 2013-03-20
=
=
<220>
<221> MISC FEATURE
= <222> (33)..(33)
<223> residue 33 is amidated
<400> 18
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ala
<210> 19
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is Aib
<400> 19
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Gly
<210> 20
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> Xaa is Aib
<220>
<221> MISC FEATURE
<222> (33)..(33)
<223> residue 33 is amidated
<400> 20
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
101
CA 02648936 2013-03-20
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Gly
<210> 21
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is Aib
<400> 21
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ser Gly
<210> 22
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is Aib
<220>
<221> MISC FEATURE
<222> (34)..(34)
<223> Residue 34 is amidated
<400> 22
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ser Gly
<210> 23
<211> 35
<212> PRT
<213> Artificial Sequence
102
CA 02648936 2013-03-20
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is Aib
<400> 23
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ser Gly Gly
<210> 24
<211> 35
<212> PRT
<213> Artificial Sequence
, <220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is Aib
<220>
<221> MISC FEATURE
<222> (35)..(35)
<223> residue 35 is amidated
<400> 24
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ser Gly Gly
<210> 25
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (2)..(2)
<223> Xaa is Aib
103
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is Aib
<400> 25
His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ser Gly
<210> 26
<211> 35
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is Aib
<400> 26
His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ser Gly Gly
<210> 27
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (2)..(2)
<223> Xaa is Aib
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is Aib
104
CA 02648936 2013-03-20
<400> 27
His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Gly
20 25 30
Cys
<210> 28
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is Aad
<400> 28
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ser Gly
<210> 29
<211> 35
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (1)..(1)
<223> Xaa at position 1 is: L-histidine, D-histidine,
desamino-histidine, 2-amino-histidine, 3-hydroxy-histidine,
homohistidine, a-fluoromethyl-histidine or a-methyl-histidine
<220>
<221> MISC FEATURE
<222> (2)..(2)
<223> Xaa at position 2 is Gly, bAla (2-aminopropionic acid),
1-amino-cylcopentanecarboxylic acid, 2-aminoisobutryic acid or an
alpha-alpha-disubstituted amino acid
<220>
<221> MISC FEATURE
<222> (3)..(3)
<223> Xaa at position 3 is Glu, Asp, or Lys
105
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (4)..(4)
<223> Xaa at position 4 is Gly or His
<220>
<221> MISC FEATURE
<222> (5)..(5)
<223> Xaa at position 5 is Thr, Ala, Gly, Ser, Leu, Ile, Val, Glu, Asp,
or Lys
<220>
<221> MISC FEATURE
<222> (6)..(6)
<223> Xaa at position 6 is: His, Trp, Phe, or Tyr
<220>
<221> MISC FEATURE
<222> (7)..(7)
<223> Xaa at position 7 is Thr or Gly
<220>
<221> MISC FEATURE
<222> (8)..(8)
<223> Xaa at position 8 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp,
or Lys
<220>
<221> MISC FEATURE
<222> (9)..(9)
<223> Xaa at position 9 is Asp or Glu
<220>
<221> MISC FEATURE
<222> (10)..(10)
<223> Xaa at position 10 is Val, Ala, Gly, Ser, Thr, Leu, Ile, Tyr,
Glu, Asp, Trp, or Lys
<220>
<221> MISC FEATURE
<222> (11)..(11)
<223> Xaa at position 11 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu,
Asp, or Lys
<220>
<221> MISC FEATURE
<222> (12)..(12)
<223> Xaa at position 12 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu,
Asp, Trp, Tyr, Lys, Homolysine, Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid, alpha, gamma-diaminobutyric acid, or homoglutamic acid
<220>
<221> MISC FEATURE
106
CA 02648936 2013-03-20
<222> (13)..(13)
<223> Xaa at position 13 is Tyr, Phe, Trp, Glu, Asp, Gin, Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic
acid, beta-Homoglutamic acid, alpha, gamma-diaminobutyric acid, or
homoglutamic acid
<220>
<221> MISC FEATURE
<222> (14)..(14)
<223> Xaa at position 14 is Leu, Ala, Gly, Ser, Thr, Ile, Val, Glu,
Asp, Met, Trp, Tyr, Lys, Homolysine, Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, alpha,
gamma-diaminobutyric acid, or homoglutamic acid
<220>
<221> MISC_FEATURE
<222> (15)..(15)
<223> Xaa at position 15 is Glu, Asp, Lys, Homolysine, Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid, alpha, gamma-diaminobutyric acid, or homoglutamic acid
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> Xaa at position 16 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu,
Asp, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine,
beta-glutamic acid, beta-Homoglutamic acid, alpha,
gamma-diaminobutyric acid, or homoglutamic acid
<220>
<221> MISC_FEATURE
<222> (17)..(17)
<223> Xaa at position 17 is Gln, Asn, Arg, Glu, Asp, Lys, Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid, alpha, gamma-diaminobutyric acid, or homoglutamic acid
<220>
<221> MISC_FEATURE
<222> (18)..(18)
<223> Xaa at position 18 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Arg,
Glu, Asp, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine,
beta-glutamic acid, beta-Homoglutamic acid, alpha,
gamma-diaminobutyric acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (19)7.(19)
<223> Xaa at position 19 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu,
Asp, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine,
beta-glutamic acid, beta-Homoglutamic acid, alpha,
gamma-diaminobutyric acid, or homoglutamic acid
107
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa at position 20 is Lys, Homolysine, Arg, Gin, Glu, Asp, His,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, alpha,
gamma-diaminobutyric acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (21)..(21)
<223> Xaa at position 21 is Leu, Glu, Asp, Lys, Homolysine, Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid, alpha, gamma-diaminobutyric acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (22)..(22)
<223> Xaa at position 22 is Phe, Trp, Asp, Glu, Lys, Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid,
beta-Homoglutamic acid, alpha, gamma-diaminobutyric acid, or
homoglutamic acid
<220>
<221> MISC FEATURE
<222> (23)7.(23)
<223> Xaa at position 23 is Ile, Leu, Val, Ala, Phe, Asp, Glu, Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic
acid, beta-Homoglutamic acid, alpha, gamma-diaminobutyric acid,
or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (24)7.(24)
<223> Xaa at position 24 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu,
Asp, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine,
beta-glutamic acid, beta-Homoglutamic acid, alpha,
gamma-diaminobutyric acid, or homoglutamic acid
<220>
<221> MISC FEATURE
<222> (25)..(25)
<223> Xaa at position 25 is Trp, Phe, Tyr, Glu, Asp, or Lys
<220>
<221> MISC FEATURE
<222> (26)..(26)
<223> Xaa at position 26 is Leu, Gly, Ala, Ser, Thr, Ile, Val, Glu,
Asp, or Lys
<220>
<221> MISC FEATURE
<222> (27)..(27)
<223> Xaa at position 27 is Val, Gly, Ala, Ser, Thr, Leu, Ile, Glu,
Asp, or Lys
108
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (28)..(28)
<223> Xaa at position 28 is Asn, Lys, Arg, Glu, Asp, or His
<220>
<221> MISC FEATURE
<222> (29)..(29)
<223> Xaa at position 29 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu,
Asp, or Lys
<220>
<221> MISC FEATURE
<222> (30)7.(30)
<223> Xaa at position 30 is Gly, Arg, Lys, Glu, Asp, or His
<220>
<221> MISC FEATURE
<222> (31)7.(31)
<223> Xaa at position 31 is Pro, Gly, Ala, Ser, Thr, Leu, Ile, Val,
Glu, Asp, or Lys
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa at position 32 is Gly, Ser, Lys, Cys, or is omitted
<220>
<221> MISC FEATURE
<222> (33)..(33)
<223> Xaa at position 33 is Gly, Ala, Ser, Thr, Ile, Val, Leu, Phe,
Pro, Cys or is omitted
<220>
<221> MISC FEATURE
<222> (34)7.(34)
<223> Xaa at position 34 is Gly, Cys, or is omitted
<220>
<221> MISC FEATURE
<222> (35)..(35)
<223> Xaa at position 35 is Gly or is omitted
<220>
<221> MISC FEATURE
<222> (35)7.(35)
<223> Residue 35 is attached to an carboxy group which is attached to
an R1 group, wherein, R1 is 0R2 or NR2R3; R2 and R3 are
independently hydrogen or (C1-C8)alkyl
<400> 29
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 30
Xaa Xaa Xaa
109
CA 02648936 2013-03-20
<210> 30
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Residue 31 is amidated
<400> 30
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 31
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (33)¨(33)
<223> Residue 33 is amidated
<400> 31
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ala
<210> 32
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (34)..(34)
<223> Residue 34 is amidated
<400> 32
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
110
CA 02648936 2013-03-20
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ser Gly
<210> 33
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys and is amidated
<400> 33
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Glu Tyr Leu Glu Lys
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 34
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys and is amidated
<400> 34
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Glu Leu Glu Gly
1 5 10 15
Lys Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 35
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)7.(32)
<223> Xaa is Lys, Orn, or Cys and is amidated
111
CA 02648936 2013-03-20
<400> 35
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Glu Glu Gly
1 5 10 15
Gin Lys Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 36
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys and is amidated
<400> 36
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Lys Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 37
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)7.(32)
<223> Xaa is Lys, Orn, or Cys and is amidated
<400> 37
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 38
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
112
CA 02648936 2013-03-20
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys and is amidated
<400> 38
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Glu Ala Ala Lys Lys Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 39
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys and is amidated
<400> 39
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Glu Ala Lys Glu Lys Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 40
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys and is amidated
<400> 40
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Glu Lys Glu Phe Lys Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 41
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
113
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys and is amidated
<400> 41
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Glu Glu Phe Ile Lys Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 42
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys and is amidated
<400> 42
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 43
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is Aad
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> residue 31 is amidated
<400> 43
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
114
CA 02648936 2013-03-20
<210> 44
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (16)7.(16)
<223> Xaa is Aad
<220>
<221> MISC FEATURE
<222> (33)..(33)
<223> Xaa is amidated
<400> 44
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ala
<210> 45
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is Aad
<220>
<221> MISC FEATURE
<222> (34)..(34)
<223> Xaa is amidated
<400> 45
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ser Gly
<210> 46
<211> 32
<212> PRT
<213> Artificial Sequence
115
CA 02648936 2013-03-20
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (12)..(12)
<223> Xaa is Aad
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys and is amidated
<400> 46
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Xaa Tyr Leu Glu Lys
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 47
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (13)..(13)
<223> Xaa is Aad
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys and is amidated
<400> 47
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Xaa Leu Glu Gly
1 5 10 15
Lys Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 48
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (14)7.(14)
<223> Xaa is Aad
116
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Urn, or Cys and is amidated
<400> 48
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Xaa Glu Gly
1 5 10 15
Gin Lys Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 49
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (15)..(15)
<223> Xaa is Aad
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Urn, or Cys, and is amidated
<400> 49
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Xaa Gly
1 5 10 15
Gin Ala Lys Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 50
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (15)..(15)
<223> Xaa is Aad
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
117
CA 02648936 2013-03-20
<400> 50
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Xaa Gin
1 5 10 15
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 51
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (17)..(17)
<223> Xaa is Aad
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 51
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Xaa Ala Ala Lys Lys Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 52
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (18)..(18)
<223> Xaa is Aad
<220>
<221> MISC FEATURE
<222> (32)7.(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 52
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Xaa Ala Lys Glu Lys Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
118
CA 02648936 2013-03-20
<.
<210> 53
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (19)..(19)
<223> Xaa is Aad
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 53
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Xaa Lys Glu Phe Lys Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 54
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is Aad
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 54
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Xaa Glu Phe Ile Lys Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 55
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
119
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (15)..(15)
<223> Xaa is Aad
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 55
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Xaa Gly
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 56
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is Aad
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> residue 31 is amidated
<400> 56
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Lys
1 5 10 15
Gin Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 57
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is Aad
<220>
<221> MISC FEATURE
120
CA 02648936 2013-03-20
<222> (33)..(33)
<223> Residue 33 is amidated
<400> 57
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Lys
1 5 10 15
Gin Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ala
<210> 58
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is Aad
<220>
<221> MISC FEATURE
<222> (34)..(34)
<223> Residue 34 is amidated
<400> 58
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Lys
1 5 10 15
Gin Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ser Gly
<210> 59
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is Aad
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
121
CA 02648936 2013-03-20
4,
<400> 59
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Lys Tyr Leu Glu Xaa
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 60
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (17)..(17)
<223> Xaa is Aad
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 60
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Lys Leu Glu Gly
1 5 10 15
Xaa Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 61
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (18)..(18)
<223> Xaa is Aad
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 61
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Lys Glu Gly
1 5 10 15
Gin Xaa Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
122
CA 02648936 2013-03-20
<210> 62
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (19)..(19)
<223> Xaa is Aad
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 62
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Lys Gly
1 5 10 15
Gin Ala Xaa Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 63
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is Aad
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 63
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Lys
1 5 10 15
Gin Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 64
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
123
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (21)..(21)
<223> Xaa is Aad
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 64
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Lys Ala Ala Lys Xaa Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 65
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (22)..(22)
<223> Xaa is Aad
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 65
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Lys Ala Lys Glu Xaa Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 66
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (23)..(23)
<223> Xaa is Aad
<220>
<221> MISC FEATURE
124
CA 02648936 2013-03-20
<222> (32)..(32)
<223> Xaa is Lys, Urn, or Cys, and is amidated
<400> 66
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Lys Lys Glu Phe Xaa Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 67
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (24)..(24)
<223> Xaa is Aad
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Urn, or Cys, and is amidated
<400> 67
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Xaa Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 68
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is Aad
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Urn, or Cys, and is amidated
<400> 68
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Lys Gly
1 5 10 15
125
CA 02648936 2013-03-20
Gin Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 69
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (30)..(30)
<223> Residue 30 is amidated
<400> 69
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gin Ala Ala Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 70
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Residue 32 is amidated
<400> 70
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gin Ala Ala Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys Ala
20 25 30
<210> 71
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC_FEATURE
<222> (33)..(33)
<223> Residue 33 is amidated
126
CA 02648936 2013-03-20
<400> 71
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gin Ala Ala Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys Ser
20 25 30
Gly
<210> 72
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Urn, or Cys, and is amidated
<400> 72
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Glu Tyr Leu Glu Gln
1 5 10 15
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 73
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Urn, or Cys, and is amidated
<400> 73
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Glu Leu Glu Gly
1 5 10 15
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 74
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
127
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 74
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Glu Glu Gly
1 5 10 15
Gin Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 75
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 75
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 76
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 76
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gin Ala Ala Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 77
<211> 31
<212> PRT
<213> Artificial Sequence
128
CA 02648936 2013-03-20
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 77
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Glu Ala Ala Lys Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 78
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 78
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Glu Ala Lys Glu Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 79
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 79
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Glu Lys Glu Phe Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 80
<211> 31
129
CA 02648936 2013-03-20
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 80
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Ala Glu Glu Phe Ile Trp Leu Val Lys Gly Arg Gly Xaa
=
20 25 30
<210> 81
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 81
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Ala Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 82
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (30)..(30)
<223> Residue 30 is amidated
<400> 82
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gin
1 5 10 15
Ala Ala Glu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
130
CA 02648936 2013-03-20
<210> 83
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Residue 32 is amidated
<400> 83
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gln
1 5 10 15
Ala Ala Glu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys Ala
20 25 30
<210> 84
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (33)..(33)
<223> Residue 33 is amidated
<400> 84
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gln
1 5 10 15
Ala Ala Glu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys Ser
20 25 30
Gly
<210> 85
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 85
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Tyr Leu Glu Glu Gln
1 5 10 15
131
I
CA 02648936 2013-03-20
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 86
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 86
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Leu Glu Gly Glu
1 5 10 15
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 87
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 87
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Glu Gly Gln
1 5 10 15
Glu Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 88
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
132
CA 02648936 2013-03-20
<400> 88
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Gly Gin
1 5 10 15
Ala Glu Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 89
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 89
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gln
1 5 10 15
Ala Ala Glu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 90
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 90
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 91
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
133
11
CA 02648936 2013-03-20
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 91
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Lys Glu Glu Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 92
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 92
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Lys Glu Phe Glu Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 93
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 93
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Ala Glu Phe Ile Glu Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 94
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
134
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 94
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Gly Gin
1 5 10 15
Ala Ala Glu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 95
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (30)..(30)
<223> Residue 30 is amidated
<400> 95
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Asp
1 5 10 15
Gin Ala Ala Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 96
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Residue 32 is amidated
<400> 96
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Asp
1 5 10 15
Gin Ala Ala Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys Ala
20 25 30
<210> 97
<211> 33
<212> PRT
<213> Artificial Sequence
135
CA 02648936 2013-03-20
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (33)..(33)
<223> Residue 33 is amidated
<400> 97
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Asp
1 5 10 15
Gin Ala Ala Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys Ser
20 25 30
Gly
<210> 98
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)T.(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 98
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Asp Tyr Leu Glu Gin
1 5 10 15
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 99
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 99
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Asp Leu Glu Gly
1 5 10 15
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
136
CA 02648936 2013-03-20
<210> 100
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Urn, or Cys, and is amidated
<400> 100
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Asp Glu Gly
1 5 10 15
Gln Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 101
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 101
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Asp Gly
1 5 10 15
Gln Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 102
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Urn, or Cys, and is amidated
<400> 102
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Asp
1 5 10 15
137
CA 02648936 2013-03-20
Gin Ala Ala Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa .
20 25 30
<210> 103
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 103
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Asp Ala Ala Lys Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 104
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 104
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Asp Ala Lys Glu Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 105
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
138
CA 02648936 2013-03-20
<400> 105
His Gly Glu Gly Thr She Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Asp Lys Glu She Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 106
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 106
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Ala Asp Glu She Ile Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 107
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (30)..(30)
<223> Residue 30 is amidated
<400> 107
His Gly Glu Gly Thr She Thr Ser Asp Val Ser Ser Tyr Leu Glu Gin
1 5 10 15
Ala Ala Asp Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 108
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
139
CA 02648936 2013-03-20
<222> (32)..(32)
<223> Residue 32 is amidated
<400> 108
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gin
1 5 10 15
Ala Ala Asp Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys Ala
20 25 30
<210> 109
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (33)..(33)
<223> Residue 33 is amidated
<400> 109
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gin
1 5 10 15
Ala Ala Asp Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys Ser
20 25 30
Gly
<210> 110
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 110
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Tyr Leu Glu Asp Gin
1 5 10 15
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 111
<211> 31
<212> PRT
<213> Artificial Sequence
140
CA 02648936 2013-03-20
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 111
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Leu Glu Gly Asp
1 5 10 15
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 112
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 112
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Glu Gly Gin
1 5 10 15
Asp Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 113
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 113
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Gly Gin
1 5 10 15
Ala Asp Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 114
<211> 31
141
CA 02648936 2013-03-20
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 114
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gin
1 5 10 15
Ala Ala Asp Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 115
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 115
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Ala Ala Lys Asp Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 116
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 116
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Lys Glu Asp Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
142
CA 02648936 2013-03-20
<210> 117
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 117
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
15
Gin Ala Lys Glu Phe Asp Ala Trp Leu Val Lys Gly Arg Gly Xaa
25 30
<210> 118
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 118
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Ala Glu Phe Ile Asp Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 119
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 119
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
143
CA 02648936 2013-03-20
Gin Ala Ala Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 120
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Urn, or Cys, and is amidated
<400> 120
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Gly Gin
1 5 10 15
Ala Ala Glu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 121
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Urn, or Cys, and is amidated
<400> 121
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 122
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
= <221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Urn, or Cys, and is amidated
144
CA 02648936 2013-03-20
<400> 122
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Lys Gly
1 5 10 15
Gin Ala Ala Glu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 123
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (15)..(15)
<223> Xaa is Aad
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 123
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Xaa Gly
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 124
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is Aad
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 124
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Lys Gly
1 5 10 15
Gin Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
145
CA 02648936 2013-03-20
<210> 125
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 125
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Asp Gly
1 5 10 15
Gin Ala Ala Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 126
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 126
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Gly Gln
1 5 10 15
Ala Ala Asp Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 127
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 127
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Asp Gly
1 5 10 15
146
CA 02648936 2013-03-20
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 128
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 128
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Lys Gly
1 5 10 15
Gin Ala Ala Asp Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 129
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (15)..(15)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 129
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Xaa Gly
1 5 10 15
Gin Ala Ala Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 130
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
147
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (19)..(19)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 130
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Gly Gin
1 5 10 15
Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 131
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (15)..(15)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 131
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Xaa Gly
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 132
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
148
CA 02648936 2013-03-20
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 132
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Lys Gly
1 5 10 15
Gin Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 133
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (15)..(15)
<223> Xaa is beta-glutamic acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Urn, or Cys, and is amidated
<400> 133
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Xaa Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 134
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is beta-glutamic acid
<220>
<221> MISC FEATURE
<222> (32)7.(32)
<223> Xaa is Lys, Urn, or Cys, and is amidated
<400> 134
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Lys Gly
1 5 10 15
149
CA 02648936 2013-03-20
Gin Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 135
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Residue 31 is amidated
<400> 135
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Asp
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 136
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (33)..(33)
<223> Residue 33 is amidated
<400> 136
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Asp
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ala
<210> 137
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (34)..(34)
<223> Residue 34 is amidated
150
CA 02648936 2013-03-20
<400> 137
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Asp
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ser Gly
<210> 138
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 138
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Asp Tyr Leu Glu Lys
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 139
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 139
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Asp Leu Glu Gly
1 5 10 15
Lys Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 140
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
151
CA 02648936 2013-03-20
<220>
<221> MISC_FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 140
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Asp Glu Gly
1 5 10 15
Gin Lys Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 141
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 141
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Asp Gly
1 5 10 15
Gin Ala Lys Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 142
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 142
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Asp
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 143
<211> 32
<212> PRT
<213> Artificial Sequence
152
CA 02648936 2013-03-20
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)T.(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 143
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Asp Ala Ala Lys Lys Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 144
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 144
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Asp Ala Lys Glu Lys Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 145
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 145
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Asp Lys Glu Phe Lys Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 146
<211> 32
153
CA 02648936 2013-03-20
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Urn, or Cys, and is amidated
<400> 146
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Asp Glu Phe Ile Lys Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 147
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Residue 31 is amidated
<400> 147
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Lys
1 5 10 15
Gln Ala Ala Asp Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 148
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (33)..(33)
<223> Residue 33 is amidated
<400> 148
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Lys
1 5 10 15
Gln Ala Ala Asp Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ala
154
CA 02648936 2013-03-20
<210> 149
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (34)..(34)
<223> Residue 34 is amidated
<400> 149
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Lys
1 5 10 15 =
Gin Ala Ala Asp Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ser Gly
<210> 150
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 150
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Lys Tyr Leu Glu Asp
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 151
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 151
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Lys Leu Glu Gly
1 5 10 15
155
CA 02648936 2013-03-20
Asp Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 152
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 152
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Lys Glu Gly
1 5 10 15
Gln Asp Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 153
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 153
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Lys Gly
1 5 10 15
Gin Ala Asp Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 154
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
156
CA 02648936 2013-03-20
<400> 154
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Lys
1 5 10 15
Gin Ala Ala Asp Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 155
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
=
<400> 155
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Lys Ala Ala Lys Asp Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 156
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 156
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Lys Ala Lys Glu Asp Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 157
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
157
CA 02648936 2013-03-20
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 157
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Lys Lys Glu Phe Asp Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 158
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 158
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Asp Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 159
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Residue 31 is amidated
<400> 159
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Lys
1 5 10 15
Gln Ala Ala Glu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 160
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
158
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (33)..(33)
<223> Residue 33 is amidated
<400> 160
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Lys
1 5 10 15
Gin Ala Ala Glu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ala
<210> 161
<211> 34
<212> PET
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (34)..(34)
<223> Residue 34 is amidated
<400> 161
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Lys
1 5 10 15
Gin Ala Ala Glu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ser Gly
<210> 162
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 162
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Lys Tyr Leu Glu Glu
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 163
<211> 32
159
CA 02648936 2013-03-20
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 163
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Lys Leu Glu Gly
1 5 10 15
Glu Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 164
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 164
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Lys Glu Gly
1 5 10 15
Gln Glu Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 165
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 165
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Lys Ely
1 5 10 15
Gin Ala Glu Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
160
CA 02648936 2013-03-20
<210> 166
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC_FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 166
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Lys
1 5 10 15
Gin Ala Ala Glu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 167
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 167
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Lys Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 168
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 168
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
161
CA 02648936 2013-03-20
Gin Lys Ala Lys Glu Glu Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 169
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC_FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 169
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Lys Lys Glu Phe Glu Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 170
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 170
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Glu Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 171
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (13)..(13)
<223> Xaa is 4-carboxy-phenyl-alanine
162
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 171
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Xaa Leu Glu Gly
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 172
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 172
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Lys Leu Glu Gly
1 5 10 15
Gin Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 173
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (13)..(13)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
163
CA 02648936 2013-03-20
<400> 173
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Xaa Leu Glu Gly
1 5 10 15
Gin Ala Ala Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 174
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (19)..(19)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 174
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Leu Glu Gly Gin
1 5 10 15
Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 175
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> Xaa is beta-glutamic acid
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Residue 31 is amidated
<400> 175
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
164
CA 02648936 2013-03-20
<210> 176
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is beta-glutamic acid
<220>
<221> MISC FEATURE
<222> (33)7-(33)
<223> Redisue 33 is amidated
<400> 176
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ala
<210> 177
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is beta-glutamic acid
<220>
<221> MISC FEATURE
<222> (34)..(34)
<223> Residue 34 is amidated
<400> 177
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ser Gly
<210> 178
<211> 32
<212> PRT
<213> Artificial Sequence
165
CA 02648936 2013-03-20
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (12)..(12)
<223> Xaa is beta-glutamic acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 178
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Xaa Tyr Leu Glu Lys
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 179
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (13)..(13)
<223> Xaa is beta-glutamic acid
<220>
<221> MISC FEATURE
<222> (32)7.(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 179
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Xaa Leu Glu Gly
1 5 10 15
Lys Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 180
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (14)..(14)
<223> Xaa is beta-glutamic acid
166
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 180
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Xaa Glu Gly
1 5 10 15
Gin Lys Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 181
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (15)..(15)
<223> Xaa is beta-glutamic acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 181
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Xaa Gly
1 5 10 15
Gin Ala Lys Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 182
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> Xaa is beta-glutamic acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
167
CA 02648936 2013-03-20
<400> 182
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 183
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (17)..(17)
<223> Xaa is beta-glutamic acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 183
His Gly Glu Gly Thr Phé Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15 =
Xaa Ala Ala Lys Lys Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 184
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (18)..(18)
<223> Xaa is beta-glutamic acid
<220>
<221> MISC_FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 184
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Xaa Ala Lys Glu Lys Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
168
CA 02648936 2013-03-20
<210> 185
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (19)..(19)
<223> Xaa is beta-glutamic acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 185
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Xaa Lys Glu Phe Lys Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 186
<211> 32
<212> PET
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is beta-glutamic acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 186
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Ala Xaa Glu Phe Ile Lys Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 187
<211> 32
<212> PET
<213> Artificial Sequence
<220>
<223> Synthetic
169
CA 02648936 2013-03-20
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> Xaa is beta-glutamic acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 187
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Lys Tyr Leu Glu Xaa
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 188
<211> 31
<212> PET
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is beta-glutamic acid
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Residue 31 is amidated
<400> 188
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Lys
1 5 10 15
Gin Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 189
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is beta-glutamic acid
<220>
<221> MISC FEATURE
170
CA 02648936 2013-03-20
<222> (33)..(33)
<223> Residue 33 is amidated
<400> 189
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Lys
1 5 10 15
Gin Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ala
<210> 190
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is beta-glutamic acid
<220>
<221> MISC FEATURE
<222> (34)..(34)
<223> Residue 34 is amidated
<400> 190
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Lys
1 5 10 15
Gin Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ser Gly
<210> 191
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (17)..(17)
<223> Xaa is beta-glutamic acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Urn, or Cys, and is amidated
171
CA 02648936 2013-03-20
f
<400> 191
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Lys Leu Glu Gly
1 5 10 15
Xaa Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 192
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (18)..(18)
<223> Xaa is beta-glutamic acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 192
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Lys Glu Gly
1 5 10 15
Gln Xaa Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 193
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (19)..(19)
<223> Xaa is beta-glutamic acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 193
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Lys Gly
1 5 10 15
Gln Ala Xaa Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
172
CA 02648936 2013-03-20
r.
<210> 194
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is beta-glutamic acid
<220>
<221> MISC_FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 194
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Lys
1 5 10 15
Gin Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 195
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (21)..(21)
<223> Xaa is beta-glutamic acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 195
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Lys Ala Ala Lys Xaa Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 196
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
173
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (22)..(22)
<223> Xaa is beta-glutamic acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 196
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Lou Glu Gly
1 5 10 15
Gin Lys Ala Lys Glu Xaa Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 197
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (23)..(23)
<223> Xaa is beta-glutamic acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 197
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Lys Lys Glu Phe Xaa Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 198
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (24)..(24)
<223> Xaa is beta-glutaimc acid
<220>
<221> MISC FEATURE
174
CA 02648936 2013-03-20
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 198
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Xaa Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 199
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Residue 31 is amidated
<400> 199
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 200
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (33)7.(33)
<223> residue 33 is amidated
<400> 200
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
175
CA 02648936 2013-03-20
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ala
<210> 201
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (34)..(34)
<223> Residue 34 is amidated
<400> 201
His Gly Glu Gly Thr She Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ser Gly
<210> 202
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (12)..(12)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 202
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Xaa Tyr Leu Glu Lys
1 5 10 15
Gin Ala Ala Lys Glu She Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
176
CA 02648936 2013-03-20
<210> 203
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (13)..(13)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 203
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Xaa Leu Glu Gly
1 5 10 15
Lys Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 204
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (14)..(14)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 204
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Xaa Glu Gly
1 5 10 15
Gln Lys Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 205
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
177
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (15)..(15)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 205
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Xaa Gly
1 5 10 15
Gin Ala Lys Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 206
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 206
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 207
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (17)..(17)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
178
CA 02648936 2013-03-20
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 207
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Xaa Ala Ala Lys Lys Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 208
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (18)..(18)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 208
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Xaa Ala Lys Glu Lys Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 209
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (19)..(19)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 209
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
179
CA 02648936 2013-03-20
Gin Ala Xaa Lys Glu Phe Lys Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 210
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 210
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Ala Xaa Glu Phe Ile Lys Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 211
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 211
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Lys Tyr Leu Glu Xaa
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 212
<211> 31
180
CA 02648936 2013-03-20
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Residue 31 is amidated
<400> 212
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Lys
1 5 10 15
Gln Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 213
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (33)..(33)
<223> Residue 33 is amidated
<400> 213
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Lys
1 5 10 15
Gln Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ala
<210> 214
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
181
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (34)..(34)
<223> Residue 34 is amidated
<400> 214
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Lys
1 5 10 15
Gin Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ser Gly
<210> 215
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (17)..(17)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 215
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Lys Leu Glu Gly
1 5 10 15
Xaa Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 216
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (18)..(18)
<223> Xaa is 4-carboxy-phenyl-alanine
182
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 216
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Lys Glu Gly
1 5 10 15
Gin Xaa Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 217
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (19)..(19)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 217
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Lys Gly
1 5 10 15
Gin Ala Xaa Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 218
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (32),.(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
183
CA 02648936 2013-03-20
<400> 218
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Lys
1 5 10 15
Gin Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 219
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (21)..(21)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 219
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Lys Ala Ala Lys Xaa Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 220
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (22)..(22)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 220
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Lys Ala Lys Glu Xaa Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
184
CA 02648936 2013-03-20
<210> 221
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (23)7.(23)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 221
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Lys Lys Glu Phe Xaa Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 222
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (24)..(24)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 222
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Xaa Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 223
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
185
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (30)..(30)
<223> Residue 30 is amidated
<400> 223
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu x4a.
1 5 10 15
Gin Ala Ala Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 224
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC_FEATURE
<222> (32)..(32)
<223> Residue 32 is amidated
<400> 224
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gin Ala Ala Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys Ala
20 25 30
<210> 225
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
186
CA 02648936 2013-03-20
<222> (33)..(33)
<223> Residue 33 is amidated
<400> 225
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gin Ala Ala Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys Ser
20 25 30
Gly
<210> 226
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (12)..(12)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 226
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Xaa Tyr Leu Glu Gin
1 5 10 15
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 227
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (13)..(13)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 227
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Xaa Leu Glu Gly
1 5 10 15
187
CA 02648936 2013-03-20
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 228
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (14)..(14)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 228
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Xaa Glu Gly
1 5 10 15
Gln Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 229
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (15)¨(15)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 229
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Xaa Gly
1 5 10 15
Gln Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 230
<211> 31
188
CA 02648936 2013-03-20
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 230
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gln Ala Ala Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 231
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (17)..(17)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 231
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Xaa Ala Ala Lys Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 232
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
189
CA 02648936 2013-03-20
=
<220>
<221> MISC FEATURE
<222> (18)..(18)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 232
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Xaa Ala Lys Glu Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 233
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (19)..(19)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 233
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Xaa Lys Glu Phe Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 234
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
190
CA 02648936 2013-03-20
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 234
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Ala Xaa Glu Phe Ile Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 235
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (15)..(15)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 235
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Tyr Leu Glu Xaa Gin
1 5 10 15
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 236
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (19)..(19)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (30)..(30)
<223> Residue 30 is amidated
<400> 236
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gin
1 5 10 15
191
CA 02648936 2013-03-20
Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 237
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (19)..(19)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Residue 32 is amidated
<400> 237
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gln
1 5 10 15
Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys Ala
20 25 30
<210> 238
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (19)..(19)
<223> Xaa is 4-carboxy-phenyl-alanine
<400> 238
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gln
1 5 10 15
Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys Ser
20 25 30
Gly
<210> 239
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
192
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 239
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Leu Glu Gly Xaa
1 5 10 15
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 240
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (17)..(17)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 240
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Glu Gly Gln
1 5 10 15
Xaa Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 241
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (18)..(18)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
193
CA 02648936 2013-03-20
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 241
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Gly Gin
1 5 10 15
Ala Xaa Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa =
20 25 30
<210> 242
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (19)..(19)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 242
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gln
1 5 10 15
Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 243
<211> 31
<212> PET
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 243
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
194
CA 02648936 2013-03-20
Ala Ala Lys Xaa Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 244
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (21)..(21)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 244
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Lys Glu Xaa Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 245
<211> 31
<212> PET
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (22)..(22)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 245
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Lys Glu Phe Xaa Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 246
<211> 31
195
CA 02648936 2013-03-20
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (23)..(23)
<223> Xaa is 4-carboxy-phenyl-alanine
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 246
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Glu Phe Ile Xaa Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 247
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 247
Ala His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
1 5 10 15
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
<210> 248
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 248
Gly His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
1 5 10 15
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
<210> 249
<211> 31
<212> PRT
<213> Artificial Sequence
196
CA 02648936 2013-03-20
<220>
<223> Synthetic
<400> 249
Pro His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
1 5 10 15
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg =
20 25 30
<210> 250
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 250
Ser His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
1 5 10 15
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
<210> 251
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 251
Thr His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
1 5 10 15
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
<210> 252
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 252
Val His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
1 5 10 15 =
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
<210> 253
<211> 32
197
CA 02648936 2013-03-20
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 253
Met Gin His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
1 5 10 15
Glu Gly Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
<210> 254
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 254
Met Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
1 5 10 15
Glu Gly Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
<210> 255
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 255
Met Lys His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
1 5 10 15
Glu Gly Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
<210> 256
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 256
Met His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
1 5 10 15
Gly Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
198
CA 02648936 2013-03-20
<210> 257
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 257
Met His His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
1 5 10 15
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
<210> 258
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 258
Met His His His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
1 5 10 15
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly
20 25 30
Arg
<210> 259
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 259
Met Tyr His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
1 5 10 15
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
<210> 260
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 260
Met Ile His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
1 5 10 15
199
CA 02648936 2013-03-20
Glu Gly Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
<210> 261
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 261
Met Asp His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
1 5 10 15
Glu Gly Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
<210> 262
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 262
Met Leu His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
1 5 10 15
Glu Gly Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
<210> 263
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 263
Met Asn His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
1 5 10 15
Glu Gly Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
<210> 264
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
200
CA 02648936 2013-03-20
<400> 264
Met Glu His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
1 5 10 15
Glu Gly Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
<210> 265
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 265
Met Trp His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
1 5 10 15
Glu Gly Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
<210> 266
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 266
Met Phe His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
1 5 10 15
Glu Gly Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
<210> 267
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 267
Met Met His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
1 5 10 15
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
<210> 268
<211> 33
<212> PRT
<213> Artificial Sequence
201
CA 02648936 2013-03-20
<220>
<223> Synthetic
<220>
<221> MISC_FEATURE
<222> (33)..(33)
<223> Residue 33 is amidated
<400> 268
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ala
<210> 269
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 269
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly
20 25 30
<210> 270
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 270
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Lys Asn Gly Gly Gly
20 25 30
<210> 271
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
202
CA 02648936 2013-03-20
<222> (30)..(30)
<223> Residue 30 is amidated
<400> 271
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly
20 25 30
<210> 272
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is Aib
<220>
<221> MISC FEATURE
<222> (30)..(30)
<223> Residue 30 is amidated
<400> 272
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Met Glu Xaa
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly
20 25 30
<210> 273
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 273
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu
20 25
<210> 274
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
203
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Residue 31 is amidated
<400> 274
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gin Ala Lys Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 275
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (34)..(34)
<223> Residue 34 is amidated
<400> 275
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gin Ala Lys Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Gly Ser
<210> 276
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (1).7(1)
<223> Xaa at position 1 is Met or omitted
<220>
<221> MISC FEATURE
<222> (2)..(2)
<223> Xaa at position 2 is Met, His, or omitted
<220>
<221> MISC FEATURE
<222> (3)..(3)
<223> Xaa at position 3 is Met, Ala, Gly, Pro, Ser, Thr, Val, Gin, Arg,
Lys, His, Tyr, Ile, Asp, Leu, Asn, Glu, Trp, or Phe
204
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (33)..(33)
<223> Residue 33 is attached to an carboxy group which is attached to
an R1 group, wherein, R1 is 0R2 or NR2R3; R2 and R3 are
independently hydrogen or (C1-C8)alkyl
<400> 276
Xaa Xaa Xaa His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Trp
1 5 10 15
Leu Glu Gly Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly
20 25 30
Arg
<210> 277
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> Residue 31 is amidated
<400> 277
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Lys Lys Glu Phe Ile Ala Trp Leu Glu Lys Gly Arg Lys
20 25 30
<210> 278
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 278
Cys Ser Gly Gly
1
<210> 279
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic
205
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is alpha, gamma-diaminobutyric acid
<400> 279
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gin Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 280
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is alpha, gamma-diaminobutyric acid
<400> 280
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gin Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ala
<210> 281
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is alpha, gamma-diaminobutyric acid
<400> 281
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ser Gly
<210> 262
<211> 32
206
CA 02648936 2013-03-20
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is alpha, gamma-diaminobutyric acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 282
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Glu Tyr Leu Glu Xaa
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 283
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic
<220>
<221> MISC FEATURE
<222> (17)7.(17)
<223> Xaa is alpha, gamma-diaminobutyric acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 283
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Glu Leu Glu Gly
1 5 10 15
Xaa Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 284
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic
207
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (18)..(18)
<223> Xaa is alpha, gamma-diaminobutyric acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 284
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Glu Glu Gly
1 5 10 15 .
Gin Xaa Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 285
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic
<220>
<221> MISC FEATURE
<222> (19)..(19)
<223> Xaa is alpha, gamma-diaminobutyric acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 285
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Xaa Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 286
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is alpha, gamma-diaminobutyric acid
<220>
<221> MISC FEATURE
208
CA 02648936 2013-03-20
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 286
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gin Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 287
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic
<220>
<221> MISC FEATURE
<222> (21)..(21)
<223> Xaa is alpha, gamma-diaminobutyric acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 287
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Glu Ala Ala Lys Xaa Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 288
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic
<220>
<221> MISC FEATURE
<222> (22)..(22)
<223> Xaa is alpha, gamma-diaminobutyric acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 288
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
209
:
CA 02648936 2013-03-20
Gin Glu Ala Lys Glu Xaa Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 289
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic
<220>
<221> MISC FEATURE
<222> (23)..(23)
<223> Xaa is alpha, gamma-diaminobutyric acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 289
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Glu Lys Glu Phe Xaa Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 290
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic
<220>
<221> MISC FEATURE
<222> (24)..(24)
<223> Xaa is alpha, gamma-diaminobutyric acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 290
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gin Ala Ala Glu Glu Phe Ile Xaa Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 291
<211> 32
210
CA 02648936 2013-03-20
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is alpha, gamma-diaminobutyric acid
<220>
<221> MISC FEATURE
<222> (32)7.(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 291
His Gly Glu Gly Thr Phe Thr Her Asp Val Her Her Tyr Leu Glu Gly
1 5 10 15
Gin Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 292
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is alpha, gamma-diaminobutyric acid
<220>
<221> MISC FEATURE
<222> (31)..(31)
<223> residue 31 is amidated
<400> 292
His Gly Glu Gly Thr Phe Thr Her Asp Val Her Her Tyr Leu Glu Xaa
1 5 10 15
Gln Ala Ala Glu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 293
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic
211
CA 02648936 2013-03-20
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> Xaa is alpha, gamma-diaminobutyric acid
<220>
<221> MISC FEATURE
<222> (33)..(33)
<223> residue 33 is amidated
<400> 293
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gin Ala Ala Glu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ala
<210> 294
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is alpha, gamma-diaminobutyric acid
<220>
<221> MISC FEATURE
<222> (34)..(34)
<223> residue 34 is amidated
<400> 294
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gln Ala Ala Glu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Ser Gly
<210> 295
<211> 32
<212> PET
<213> Artificial Sequence
<220>
<223> synthetic
<220>
<221> MISC FEATURE
<222> (12)..(12)
<223> Xaa is alpha, gamma-diaminobutyric acid
212
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 295
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Xaa Tyr Leu Glu Glu
1 5 10 15
Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 296
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic
<220>
<221> MISC FEATURE
<222> (13)..(13)
<223> Xaa is alpha, gamma-diaminobutyric acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 296
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Xaa Leu Glu Gly
1 5 10 15
Glu Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 297
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic
<220>
<221> MISC FEATURE
<222> (14)..(14)
<223> Xaa is alpha, gamma-diaminobutyric acid
<220>
<221> MISC FEATURE
<222> (32)¨(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
213
CA 02648936 2013-03-20
<400> 297
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Xaa Glu Gly
1 5 10 15
Gin Glu Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 298
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic
<220>
<221> MISC FEATURE
<222> (15)..(15)
<223> Xaa is alpha, gamma-diaminobutyric acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 298
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Xaa Gly
1 5 10 15
Gin Ala Glu Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 299
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic
<220>
<221> MISC FEATURE
<222> (16)..(16)
<223> Xaa is alpha, gamma-diaminobutyric acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 299
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa
1 5 10 15
Gin Ala Ala Glu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
214
CA 02648936 2013-03-20
<210> 300
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic
<220>
<221> MISC FEATURE
<222> (17)..(17)
<223> Xaa is alpha, gamma-diaminobutyric acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 300
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Xaa Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 301
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic
<220>
<221> MISC FEATURE
<222> (18)..(18)
<223> Xaa is alpha, gamma-diaminobutyric acid
<220>
<221> MISC_FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 301
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Xaa Ala Lys Glu Glu Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 302
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic
215
CA 02648936 2013-03-20
<220>
<221> MISC FEATURE
<222> (19)..(19)
<223> Xaa is alpha, gamma-diaminobutyric acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Urn, or Cys, and is amidated
<400> 302
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Xaa Lys Glu Phe Glu Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 303
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic
<220>
<221> MISC FEATURE
<222> (20)..(20)
<223> Xaa is alpha, gamma-diaminobutyric acid
<220>
<221> MISC FEATURE
<222> (32)..(32)
<223> Xaa is Lys, Urn, or Cys, and is amidated
<400> 303
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Xaa Glu Phe Ile Glu Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
<210> 304
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic
<220>
<221> MISC FEATURE
<222> (15)..(15)
<223> Xaa is alpha, gamma-diaminobutyric acid
<220>
<221> MISC FEATURE
216
,
CA 02648936 2013-03-20
<222> (32)..(32)
<223> Xaa is Lys, Orn, or Cys, and is amidated
<400> 304
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Xaa Gly
1 5 10 15
Gin Ala Ala Glu Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Xaa
20 25 30
217
